Molecular biology

1. 2. Motor 3. Signal sequences-navigated transport 4. Apoptosis 5. RNA: structure and function 6. RNAinterference 7. Control of gene expression 8. Cojugative gene transfer 9. DNA damage response 10. –cell signaling 11. Bacterial chemotaxis 12. Bacterial cell motility 13. Bacterial toxins 14. Viroids

Vladimír Jirků

1. Cytoskeleton

The Cytoskeleton is a highly dynamic (complex, responsive) filamentous structure, acting as an intracellular scaffolding organizing the cell's contents to maximize inner-cell differentiation, transport and coordination of cell processes, as well as maintaining or altering cell shape and cell motion. In addition, interactions with the cytoskeleton influence a number of other behaviors, including signaling pathways (But one of the most important roles of the cytoskeleton is that it regulates and thus coordinates polymerization. Without this coordination, cells can't maintain their ability to have front ends and back ends – this front / back dichotomy is referred to as “cell polarity”). It is contained in all eukaryotic cells. While the cytoskeleton is an interconnected network, it is conventionally broken into three distinct cytoplasmic systems: , actin filaments ( ) and intermediate filaments.

Microtubules:

They are hollow filaments of about 25 nm, formed by 13 protofilaments which, in turn, are polymers of alpha and beta . They have a very dynamic behaviour, binding GTP for polymerization. They are organized by the microtubule organizing centre (MTOC, , - in yeast cell). They play key roles in: intracellular transport (associated with motor proteins) - transport cell compartments; the of cilia and flagella; the mitotic spindle; synthesis of the in plant cell, among others. Microtubules have + end / - end (MTOC) polarity.

Actin filaments

These filaments (around 7 nm in diameter) are composed of two actin chains oriented in an helicoidal shape. They are mostly concentrated just beneath the plasma membrane, as they keep cellular shape, form cytoplasmatic protuberances (like pseudopodia and microvili), and participate in some cell-to-cell or cell-to-matrix junctions and in the transduction of signals. They are also important for and, along with , muscular contraction. G- actin (globular actin) with bound ATP can polymerize, to form F-actin (filamentous actin).

F-actin may hydrolyze its bound ATP to ADP + Pi and release Pi. ADP release from the filament does not occur because the cleft opening is blocked. ADP/ATP exchange: G-actin can release ADP and bind ATP, which is usually present in the at higher concentration than ADP. Actin filaments have polarity. The actin monomers all orient with their cleft toward the same end of the filament (designated the minus end). Actin monomers spiral around the axis of the filament, with a structure resembling a double helix. At the ends of actin filaments are boud capping proteins. Different capping proteins may either stabilize an actin filament or promote disassembly. They may have a role in determining filament length. Cross-linking proteins organize actin filaments into bundles or networks. Actin- binding domains of several of the cross-linking proteins (e.g., , -, , and fimbrin) are homologous. Most cross-linking proteins are dimeric or have 2 actin-binding domains. Some actin-binding proteins such as -actinin, villin and fimbrin bind actin filaments into parallel bundles. Depending on the length of a cross-linking protein, or the distance between actin-binding domains, actin filaments in parallel bundles may be held close together, or may be far enough apart to allow interaction with other proteins such as myosin. dimerize, through antiparallel association of their C-terminal domains, to form V-shaped cross-linking proteins that have a flexible shape due to hinge regions. Filamins organize actin filaments into loose networks that give some areas of the cytosol a gel-like consistency. Filamins may also have scaffolding roles relating to their ability to bind constituents of signal pathways such as plasma membrane receptors, calmodulin, caveolin, protein kinase C, transcription factors, etc. Nucleation. three 'barbed'-end nucleators of F actin have been described. The ARP2/3 complex, made up of two ARP proteins and five associated subunits (ARPC1–5/p41–16), serves as a template for F-actin formation and can also interact with the sides of existing actin filaments, forming branched F-actin arrays. Upstream regulation: cofilins are inhibited in their activity and dynamics by specific kinases (such as LIM (Lin-11/Isl-1/Mec-3) or TES kinases) that phosphorylate the proteins on the conserved serine residue. Dephosphorylation by phosphatases activates cofilins. ARP, actin-related protein; CAP, cyclase-associated protein; FH1, formin 1; FH2, formin homology

Filopodia (also called microspikes) are long, thin and transient processes that extend out from the cell surface. Bundles of parallel actin filaments, with their plus ends oriented toward the filopodial tip, are cross-linked within filopodia by a small actin-binding protein such as fascin. The closely spaced actin filaments provide stiffness.

Microvilli are shorter / more numerous protrusions of the cell surface found in some cells. Tightly bundled actin filaments within these structures also have their plus ends oriented toward the tip. Small cross-linking proteins such as fimbrin and villin bind actin filaments together within microvilli.

Lamellipodia are thin but broad projections at the edge of a mobile cell. Lamellipodia are dynamic structures, constantly changing shape. Lamellipodia, at least in some motile cells, have been shown to contain extensively branched arrays of actin filaments, oriented with their plus (barbed) ends toward the plasma membrane. Forward extension of a lamellipodium occurs by growth of actin filaments adjacent to the plasma membrane.

Cells move through the rapid rearrangement of the actin cytoskeleton. In the dendritic nucleation model, several signaling pathways converge to activate WASp/Scar proteins, which in turn activate the Arp2/3 complex. Active Arp2/3 complex binds to the side of an existing filament and nucleates new filament growth towards the . The combined force from many growing filaments pushes the cell membrane forward, moving the cell..

ARP, actin-related protein; CAP, cyclase-associated protein; FH1, formin homology 1; FH2, formin homology

Intermediate filaments

These filaments, 8 to 11 nanometers in diameter, are strongly bound and very heterogeneous constituents of the cytoskeleton. They organize the internal tridimensional structure of the cell (they are structural support of the nuclear membrane for example). They also participate in some cell-cell and cell-matrix junctions. Respectively, they are made from many different types of proteins (, , , …….). Intermediate filaments are essential for normal tissue structure and function; they provide physical resilience for cells to withstand the mechanical stresses of the tissue in which they are expressed. They are found in the nucleus () and the (cytoplasmic intermediate filaments).

Assembly: Intermediate filaments are assembled from tetramers: two monomers form a parallel dimer by the winding of their α-helical rods into a coiled coil, oriented in register and in the same direction, and then two dimers join side-by-side in a staggered anti-parallel orientation to form a bidirectional tetramer. Each dimer is 48 nm long; because the dimers are staggered the tetramer is somewhat longer. The anti-parallel orientation of tetramers means that, unlike microtubules and microfilaments (which have a preferred assembly end), intermediate filaments do not show polarized unidirectional properties. Assembly and disassembly is regulated by cycles of phosphorylation and dephosphorylation; polymerization of intermediate filaments occurs rapidly and does not require cofactors or associated proteins.

In most cells, intermediate filaments assemble into complex networks that course through the cytoplasm between the nucleus and the cell surface. Towards the cell center, they are attached to the nuclear envelope, and in the region of the plasma membrane, they are associated with various adhesion structures such as the desmosomes and hemidesmosomes of epithelial cells and the focal adhesions of fibroblasts. Cytoskeletal intermediate filaments play important roles in a wide range of cellular functions. These include the formation and maintenance of cell shape, cellular mechanical integrity, signal transduction, and the overall stability and integration of other cytoskeletal systems, i.e. microtubules and actin filaments.

Types of intermediate filaments: The proteins comprising these filasments are encoded by approximately 70 different genes. This large family of proteins is subdivided into five types, four of which are located in the cytoplasm (cytoskeletal IF) and one in the nucleus (nucleoskeletal IF). In the cytoplasm, one, two or even more types of IF protein chains can polymerize into cytoskeletal IF of 10 nm diameter. Textbooks describe IF as very stable and rigid structures, only recognized for their maintenance of the mechanical stability of cells. However, the results of live cell imaging studies demonstrate the opposite. These studies have shown that IF networks are also active and dynamic components of the cytoskeleton.

The cytoskeleton in cells (??)

The cytoskeleton is a key feature of eukaryotic cells, but homologues to the major proteins of the eukaryotic cytoskeleton have recently been found in ; FtsZ: a was the first protein of the prokaryotic cytoskeleton to be identified. Like tubulin, FtsZ forms filaments in the presence of GTP, but these filaments do not group into tubules. During , FtsZ is the first protein to move to the division site, and is essential for recruiting other proteins that produce a new cell wall between the dividing cells. MreB and ParM: Prokaryotic actin- like proteins, such as MreB, are involved in the maintenance of cell shape. All non-spherical have genes encoding actin-like proteins, and these proteins form a helical network beneath the cell membrane that guides the proteins involved in cell wall biosynthesis. Some encode a partitioning system that involves an actin-like protein ParM. Filaments of ParM exhibit dynamic instability, and may partition DNA into the dividing daughter cells by a mechanism analogous to that used by microtubules during eukaryotic mitosis.

2. Motor proteins

Eukaryotic cells —from single-celled fungi to those in humans—are equipped with a sophisticated transportation infrastructure. Motor proteins haul molecular cargo to and from different locations inside cells by traveling along a network of cytoskeleton. Classic motor proteins appear as highly assymetrical elongated molecules with a common head –stalk – tail organisation. Eaech of the globular head contains a mechanochemical domain with nukleotide – and microtubule- or actin binding sites which is higly conserved in particular groups of motor proteins. The head(s) undergo conformational changes during nucleotide (ATP) hydrolysis to produce mechanical tasks (translocation, sliding self-asembly, depolymerization). Attached to the head is a central rod-like stalk domain generally containing α-helix motifs.The stalks are unique in the various related motors, reflecting their diverse role in the cell. Located mostly at the C-terminus of the molekule is a non conserved tail with binding sites that specify the type of cargo (membrane vesicle, , protein complex) to be transported along associated microtubules or actin filaments.

There are two classes of motor proteins, and that direct organelle and particle movement along microtubules. Both are termed Microtubule Associated Proteins (MAPs). are a family of proteins, that are involved in organelle transport, in mitosis, in meiosis, and in the transport of synaptic vesicles along axons. Cytoplasmic are involved in organelle transport and mitosis. Kinesins and dyneins are myosin-like proteins composed of two heavy chains plus several light chains. Each heavy chain contains a conserved, globular, ATP-binding head and a tail composed of a string of rodlike domains. The two head domains are ATPase motors that bind to microtubules, while the tails generally bind to specific cell components and thereby specify the type of cargo that the protein transports. and vesicles containing kinesin move from the minus end of a microtubule (at a microtubule organizing center, such as centrosome) to the plus end. Hence, kinesin produces movement from the center of a cell to its periphery, called anterograd transport. In contrast, cytoplasmic dynein moves the particles from the plus end to the minus end of the microtubules , called retrograd transport.

Myosins: are a large superfamily of motor proteins that move along actin filaments, while hydrolyzing ATP.About 20 classes of myosin have been distiguished on the basis of the sequence of amino acids in their ATP-hydrolyzing motor domain. The different calsses of myosin also differ in structure of their tail domains. Tail domains have various functions in different myosin classes, inncluding dimerization and other protein interactin.The actin-based motor skeletal muscle myosin in the centre is flanked by the microtubule motors conventional kinesin on the left and cytoplasmic dynein on the right. All three motors consist of a dimer of two heavy chains whose catalytic domains are shown in yellow, whereas the stalks, which form extended coiled-coils in both myosin and kinesin, are shown in blue. Associated polypeptides (four light chains in skeletal muscle myosin, two light chains in conventional kinesin, and a complex set of intermediate, light-intermediate and light chains in dynein) are shown in purple. The 'antennae' extending from the dynein heads contain the microtubule binding site, which in myosin and kinesin is part of the compact head.

3. Signal sequences-navigated protein transport

The movement of proteins across or into a membrane is an intrinsically complex process. First, the protein destinated for translocation or integration must be targeted to the membrane. Second, protein must be physically moved across or into a membrane that normally prevent such a movement. Third, once at the membrane, secretory proteins must be distinguished from membrane proteins so that the later can be moved into the membrane bilayer, but only after being properly oriented with respekt to the plane of the membrane. Fourth, the permeability barrier of the membrane must be maintaned throughout, even though a macromolecule or a portion of it is traversing the billayer. The information that enable the cellular transport machinery to correctly position a protein inside or outside the cell is contained in the polypetide chain an is called signal sequence. Signal sequence: The essential signal for correct sorting of such proteins are hydrophobic N- terminal signal sequences typically comprising 15-20 amino acids: a short positively charged N-terminal region, a central hydrophobic core and a more polar C terminal part which has a cleavage site for signal peptidase. Signal sequences are very divergent and have two general features – hydrophobicity and α-helical conformation of the hydrophobic core. Disruption of one of these features leads to a non-functional signal sequences for cotranslational targeting. While in signal sequences are usually located at the extreme N-terminus, in prokaryotes like E. coli SRP-dependent signal sequences are often a transmembrane helix within membrane proteins of the plasma membrane.

Co-translational translocation. The N-terminal signal sequence of the protein is recognized by a signal recognition particle (SRP) while the protein is still being synthesized on the . The synthesis pauses while the ribosome-protein complex is transferred to a SRP receptor on the endoplasmic reticulm (ER). SRP will recognize any signal sequence with a critical level of hydrophobicity. The question how SRP recognizes and binds almost any hydrophobic α-helix is currently unanswered. Binding of SRP will arrest elongation of the nascent peptide chain and target the complex to the membrane via GTP dependent interaction with the SRP receptor (SR) / docking protein (DP). After the SRP-SR-nascent chain-ribosome complex interacts with the translocon, the signal sequence is released; the SRP-SR complex dissociates after GTP hydrolysis and translation resumes There, the nascent protein is inserted into the translocatin complex (translocon) that passes through the ER membrane. The signal sequence is cleaved from the polypeptide once it has been translocated into the ER by signal peptidase. This signal sequence processing differs for different tus navigated proteins. Within the ER, the protein is mostly covered by a chaperone protein to protect it from the high concentration of other proteins in the ER, giving it time to fold correctly. Once folded, the protein is modified as needed, then transported to the for further processing and movement (vesicular transport). Post-translational translocation: Even though most proteins are cotranslationally translocated, some are translated in the cytosol and later transported to their destination. This applies to mitochondrial, or nuclear proteins. The main advantage of cotranslational targeting is that the coupling of translation and translocation prevents misfolding of the proteins in the cytoplasm.

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Signal recognition particle (SRP): particle displays three main activities in the process of cotranslational targeting: (I) binding to signal sequences emerging form the translating ribosome, (II) pausing of peptide elongation, and (III) promotion of protein translocation through docking to the membrane-bound SRP receptor (FtsY in prokaryotes) and transfer of the ribosome nascent chain complex (RNC) to the protein-conducting channel. These activities can be assigned to the two main domains of SRP separable by the micrococcal nuclease treatment: the first domain, called S-domain, binds to signal sequences and promotes translocation. In mammalian SRP, it includes about half of 7S RNA of SRP (~nucleotides 100-250) as well as the essential proteins SRP19, SRP54 (Ffh in prokaryotes), and the SRP68/72 heterodimer (fig. 2, 3a). While SRP19 is required for SRP assembly, SRP54 is the functionally most significant protein subunit of the S-domain: it recognizes the signal sequence and interacts with the SRP receptor in a GTP-dependent manner. SRP54 is composed of an N-terminal domain (N), a central GTPase domain (G) and a methionine-rich C-terminal domain (M) , which anchors SRP54 to SRP RNA. In addition, together with a part of the RNA backbone, the M-domain carries out the principal function of the signal sequence recognition near the peptide tunnel exit site of the large ribosomal subunit. The second main domain of SRP, called Alu-domain, mediates the elongation arrest activity. It is supposed to enable efficient targeting by providing a time window during which the nascent chain can be targeted to the translocation site. The Alu-domain contains the 5‘- and 3‘-part of 7S RNA (including Alu-like sequences) as well as the SRP9/14 heterodimer, which is essential for its aktivity. SRP RNA: The presence and necessity of RNA in SRP is not completely understood yet. It seems that 4.5S RNA in E.coli stabilises the structure of the Ffh M-domain. In addition, kinetic studies show that RNA enhances association and dissociation of Ffh-FtsY complexes, and the positively charged N-terminal part of the signal sequence probably interacts with the negatively charged RNA backbone. The advantage of cotranslational targeting is that coupling of translation and translocation prevents misfolding of newly synthesized protein in cytoplasm. But protein translation can be faster then the diffusion of SRP-RNC complex to the membrane. To prevent that, SRP retards the translation and in that way it enlarges the time window during which a nascent chain can be targeted before it reaches a critical length prone to fold or aggregate.

Signal sequence cleavage: In eukaryotes, signal sequences direct the insertion of proteins into the membrane of the and are usually cleaved off by signal peptidase. The resulting signal peptides are presumably rapidly degraded, but some still have functions on their own. Here, we describe examples of post-targeting functions of membrane-integral signal peptides, of signal peptides released from the membrane into either the cytosol or endoplasmic reticulum lumen and of signal peptide fragments generated by intramembrane cleavage. Thus, signal peptides must be considered as an additional resource in the context of the function of secretory and membrane proteins.

Thus navigated proteins leaving the Golgi apparatus undergo vesicular transport and consequent exocytosis. This protein movement occurs constitutively. In contrast, the exocytosis of secretory proteins is a highly regulated process, in which a ligand must bind to a receptor to trigger vesicle fusion and protein secretion. Vesicular transport is thus a major cellular activity, responsible for molecular traffic between a variety of specific membrane- enclosed compartments. The selectivity of such transport is therefore key to maintaining the functional organization of the cell. For example, lysosomal enzymes must be transported specifically from the Golgi apparatus to —not to the plasma membrane or to the ER. These proteins are transported within vesicles, so the specificity of transport is based on the selective packaging of the intended cargo into vesicles that recognize and fuse only with the appropriate target membrane. Because of the central importance of vesicular transport to the organization of eukaryotic cells, understanding the molecular mechanisms that control vesicle packaging, budding, and fusion is a major area of research in . Finally, The fusion of a transport vesicle with its target involves two types of events. First, the transport vesicle must specifically recognize the correct target membrane; Second, the vesicle and target membranes must fuse, thereby delivering the contents of the vesicle to the target organelle. Research over the last several years has led to development of a model of vesicle fusion in which specific recognition between a vesicle and its target is mediated by interactions between unique pairs of transmembrane proteins, followed by fusion between the phospholipid bilayers of the vesicle and target membranes.

4. Apoptosis

Cell death is said to occur by more alternative, opposite modes: apoptosis, a programmed, managed form of cell death, necrotic death, an unordered and accidental form of cellular dying, and „physiological― death, i.e. the death cause by natural, replicative ageing The incorrect consequence is the overlapping of: a) the process whereby cells die, cell death; and b) the changes that the cells and tissues undergo after the cells die. Only the latter process can be referred to as necrosis and represents a ‗no return‘ process in cell life. In general, these pathobiological processes remain poorly understood and the physiological and biochemical factors that lead to cell death are still not clear. Our understanding of the mechanisms involved in the process of apoptosis in mammalian cells transpired from the investigation of programme cell death that occurs during the development of the nematode Caenorhabditis elegans. In this organism 1090 somatic cells are generated in the formation of the adult worm, of which 131 of these cells undergo apoptosis or ―programmed cell death.‖ These 131cells die at particular points during the development process, which is essentially invariant between worms, demonstrating the remarkable accuracy and control in this system. Apoptosis has since been recognized and accepted as a distinctive and important mode of ―programmed‖ cell death, which involves the genetically determined elimination of cells. However, it is important to note that other forms of programmed cell death have been described and other forms of programmed cell death may yet be discovered. Apoptosis occurs normally during development and aging and as a homeostatic mechanism to maintain cell populations in tissues. Apoptosis also occurs as a defense mechanism such as in immune reactions or when cells are damaged by disease or by an agent damaging DNA. Although there are a wide variety of stimuli and conditions, both physiological and pathological, that can trigger apoptosis, not all cells will necessarily die in response to the same stimulus. At low doses, a variety of injurious stimuli such as heat, radiation, hypoxia and cytotoxic anticancer drugs can induce apoptosis but these same stimuli can result in necrosis at higher doses. Finally, apoptosis is a coordinated and often energy-dependent process that involves the activation of a group of cysteine proteases called ―caspases‖ and a complex cascade of events that link the initiating stimuli to the final demise of the cell. Apoptic cell morphology / cytology: During the early process of apoptosis, cell shrinkage and pyknosis are visible by light microscopy. With cell shrinkage, the cells are smaller in size, the cytoplasm is dense and the organelles are more tightly packed. Pyknosis is the result of chromatin condensation and this is the most characteristic feature of apoptosis. Elektron microscopy can better define the subcellular changes. Early during the chromatin condensation phase, the electron-dense nuclear material characteristically aggregates peripherally under the nuclear membrane although there can also be uniformly dense nuclei. Extensive plasma membrane blebbing (below) occurs followed by separation of cell fragments into apoptotic bodies during a process called ―budding.‖ Apoptotic bodies consist of cytoplasm with tightly packed organelles with or without a nuclear fragment.

Apoptic cell biochemistry:

Apoptotic cells exhibit several biochemical modifications such as protein cleavage, protein cross-linking, DNA breakdown, and phagocytic recognition that together result in the distinctive structural pathology described previously Caspases are widely expressed in an inactive proenzyme form in most cells and once activated can often activate other procaspases, allowing initiation of a protease cascade. Some procaspases can also aggregate and autoactivate. This proteolytic cascade, in which one caspase can activate other caspases, amplifies the apoptotic signaling pathway and thus leads to rapid cell death. Caspases have proteolytic activity and are able to cleave proteins at aspartic acid residues, although different caspases have different specificities involving recognition of neighboring amino acids. Once caspases are initially activated, there seems to be an irreversible commitment towards cell death. Ten major caspases have been broadly categorized into initiators (caspase-2,-8,-9,-10), effectors or executioners (caspase-3,-6,-7) and inflammatory caspases (caspase-1,-4,-5). Extrinsic pathway: The extrinsic signaling pathways that initiate apoptosis involve transmembrane receptor-mediated interactions. The sequence of events that define the extrinsic phase of apoptosis are best characterized with the FasL/FasR and TNF-α/TNFR1 models. This pathway leads to the the auto-catalytic activation of procaspase-8. Intrinsic Pathway: The intrinsic signaling pathways that initiate apoptosis involve a diversearray of non-receptor-mediated stimuli that produce intracellular signals that act directly on targets within the cell and are mitochondrial-initiated events. The stimuli that initiate the intrinsic pathway produce intracellular signals that cause changes in the inner mitochondrial membrane that results in an opening of the mitochondrial permeability transition (MPT) pore, loss of the mitochondrial transmembrane potential and release of two main groups of normally sequestered pro-apoptotic proteins from the intermembrane space into the cytosol. The first group consists of cytochrome c, Smac/DIABLO, and the serine protease HtrA2/Omi. These proteins activate the caspasedependent mitochondrial pathway. Cytochrome c binds and activates Apaf-1 as well as procaspase-9, forming an ―apoptosome‖ The clustering of procaspase-9 in this manner leads to caspase-9 activation. The control and regulation of these apoptotic mitochondrial events occurs through members of the Bcl-2 family of proteins. The Bcl-2 family of proteins governs mitochondrial membrane permeability and can be either pro- apoptotic or antiapoptotic. Some of the anti-apoptotic proteins include Bcl-2, Bcl-x, Bcl-XL, Bcl-XS, Bcl-w, BAG, and some of the pro-apoptotic proteins include Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk. These proteins have special significance since they can determine if the cell commits to apoptosis or aborts the process. It is thought that the main mechanism of action of the Bcl-2 family of proteins is the regulation of cytochrome c release from the mitochondria via alteration of mitochondrial membrane permeability. A few possible mechanisms have been studied but none have been proven definitively. Mitochondrial I. II.

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damage in the Fas pathway of apoptosis is mediated by the caspase-8 cleavage. This is one example of the ―cross-talk‖ between the death-receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway. The extrinsic and intrinsic pathways both end at the point of the execution phase, considered the final pathway of apoptosis. It is the activation of the execution caspases that begins this phase of apoptosis. Execution caspases activate cytoplasmic endonuclease, which degrades nuclear material, and proteases that degrade the nuclear and cytoskeletal proteins. Caspase-3, caspase-6, and caspase-7 function as effector or “executioner” caspases. Caspase-3 is considered to be the most important of the executioner caspases and is activated by any of the initiator caspases (caspase-8, caspase-9, or caspase- 10). The role of apoptosis in normal physiology is as significant as that of its counterpart, mitosis. It demonstrates a complementary but opposite role to mitosis and cell proliferation in the regulation of various cell populations. It is estimated that to maintain homeostasis in the adult human body, around 10 billion cells are made each day just to balance those dying by apoptosis. And that number can increase significantly when there is increased apoptosis during normal development and aging or during disease. Alterations of various cell signaling pathways can result in dysregulation of apoptosis and lead to cancer. The p53 tumor suppressor gene is a transcription factor that regulates the cell cycle and is the most widely mutated gene in human tumorigenesis (Wang and Harris, 1997). The critical role of p53 is evident by the fact that it is mutated in over 50% of all human cancers. p53 can activate DNA repair proteins when DNA has sustained damage, can hold the cell cycle at the G1/S regulation point on DNA damage recognition, and can initiate apoptosis if the DNA damage proves to be irreparable (Pientenpol and Stewart, 2002). Tumorigenesis can occur if this system goes awry. If the p53 gene is damaged, then tumor suppression is severely reduced. The p53 gene can be damaged by radiation, various chemicals, and viruses such as the human papillomavirus (HPV). As mentioned previously, p53 then signals growth arrest of the cell at a checkpoint to allow for DNA damage repair or can cause the cell to undergo apoptosis if the damage cannot be repaired. This system can also be inactivated by a number of mechanisms including somatic genetic/epigenetic alterations and expression of oncogenic viral proteins such as the HPV.

There is evidence of other forms of non-apoptotic programmed cell death that should also be considered since they may lead to new insights into cell death programs and reveal thein potentially unique roles in development, homeostasis, neoplasia and degeneration. It has become increasingly apparent that cell death mechanisms include a highly diverse array of phenotypes and molecular mechanisms. And there is evidence that modulation of one form of cell death may lead to another. Because other types of cell death may require gene activation and function in an energy dependent manner, they are also considered to be forms of ―programmed cell death.‖ Therefore, there is some resistance to the exclusive use of the term ―programmed cell death‖ to specifically describe apoptosis.

5. RNA: structure and function

RNA secondary structure is any 3D form of this biopolymer; the secondary structure is highly important to the correct function of the RNA — often more so than the actual sequence. This fact aids in the analysis of on-coding RNA sometimes termed "RNA genes". Since it is almost entirely base pair-mediated, RNA secondary structure can be said to define which bases are paired in a molecule or complex. However, the traditional Watson-Crick base pair is not the only type of pairing that is permissible in RNA; Hoogsteen base pairing is also common. Generally, RNA secondary structure is divided into helices (contiguous base pairs), and various kinds of loops (unpaired nucleotides surrounded by helices). The stem-loop structure in which a base-paired helix ends in a short unpaired loop is extremely common and is a building block for larger structural motifs such as cloverleaf structures, which are four-helix junctions such as those found in tRNA. Internal loops (a short series of unpaired bases in a longer paired helix) and bulges (regions in which one strand of a helix has "extra" inserted bases with no counterparts in the opposite strand) are also frequent. Finally, both pseudoknots and base triples are presentas well. In addition, RNA asssociates with various proteins to form ribonukleoprotein particles (RNPs)

The key functins of RNA (gene expression process, RNA interference) require recognizable "domains" of secondary structure like hairpin loops, bulges and internal loops. In this connection, there has been a significant amount of bioinformatics research directed at the RNA structure prediction problem.

In the expression of eukaryotic structural genes can be distinguished at least seven control levels: DNA accessibility, DNA transcription (see Chapter 6.), post-transcriptional modifications of pre-mRNA (RNA capping, splicing and editing, RNA export from the nucleus, regulation of RNA stability, translation and post-translational modification of de novo synthetized protein. (Processing of mRNA differs greatly among eukaryotes, bacteria, and archea). Non-eukaryotic mRNA is essentially mature upon transcription and requires no processing, except in rare cases.)

RNA capping: A 5' cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the "front" or 5´end of a eukaryotic messenger RNA shortly after the start of transcription. The 5' cap consists of a terminal 7-methylguanosine residue which is linked through a 5'-5'-triphosphate bond to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from Rnases. This process occurs co-transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5' end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. RNA molecule is capped by the addition of a guanosine monophosphate residue (from GTP) via a 5´-5′ - pyrophosphate linkage. After addition of the cap nucleotide, a methy group is added to the N7 position of the guanine base producing a Cap 0 structure. (the addition of another methyl group onto the penultimate nucleotide from the 5′-end of the mRNA (producing a Cap 1 structure) will further boost translation - formation of an mRNA ribosome complex.) .

RNA polyadenylation: The site of processing and polyadenylation of a pre-mRNA is established by the coincidence of several distinctive cis elements in the RNA, collectively known as the polyadenylation signal. The processing complex possesses a number of RNA- binding subunits. In mammals, these include the 160 kD and 100 kD subunits of the Cleavage and Polyadenylation Specificity Factor (abbreviated as CPSF160 and CPSF100, respectively), the 64 kD subunit of Cleavage stimulatory Factor (CstF64), the 25 kD subunit of Cleavage Factor II (CFIm25), CPSF30, and Fip1. What is interesting about this collection is that there are only three components of the mammalian polyadenylation signal – the poly(A) signal AAUAAA, the downstream element, and the upstream sequence element. These three components have been associated with CPSF160, CstF64, and CFIm25, respectively. (A similar situation is seen in yeast). Fip1 has been shown to associate with the actual processing site itself. CPSF100 and CPSF30 have not been ―localized‖ to any particular part of the pre-mRNA (although the yeast counterpart of CPSF30, Yth1, has been shown to associate with the cleavage site itself, much as does the human Fip1). In mammals (bellow), the five factors involved are indicated, showing the subunit structures and molecular weights of CPSF and CstF. The polymerase II CTD (grey) is also shown. Poly(A) polymerase adds tracts of poly(A) to the 3‘ ends of RNA made by RNA polymerase. Moreover, these 3‘ ends are generated by the cutting, or processing, of the precursor mRNA.

Poly(A) tail addition is preceded by an RNA processing event, it follows that there must be a way for the responsible enzymes to identify the site of processing. It turns out that each precursor mRNA possesses an array of RNA sequence elements that direct the processing and polyadenylation complex. These elements are collectively known as the polyadenylation signal. (AAUAAA is the polyadenylation signal in mammalian mRNAs).

A poly(A) tail is found at the 3′ end of nearly every fully processed eukaryotic mRNA and has been suggested to influence some key aspects of mRNA metabolism: mRNA stability, mRNA‘s translational efficiency, and the efficiency of mRNA transport from the nucleus to the cytoplasm. There is an upper limit of 200–300 residues in the length of poly(A) tails found on newly synthesized RNA in vivo. This length restriction is mediated, in part, by poly(A)- binding protein II (PAB II), a nuclear protein with high affinity for poly(A). Once a short poly(A) tail has been synthesized, PAB II binds to it and forms a quaternary complex with CPSF, PAP, and the substrate RNA. This complex transiently stabilizes the binding of PAP to the RNA 3′ end, supporting processive synthesis of a long poly(A) tail in a single rapid step. The poly(A) tail is shortened over time.

RNA splicing: is a modification of pre-mRNA (hnRNA) in which introns are removed and exons are joined; splicing is done in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (sn RNPs). The major spliceosomal snRNPs U1, U2, U4, U5 and U6 are responsible for splicing the vast majority of pre-mRNAs (so-called U2 introns). A group of less abundant snRNPs, U11, U12, U4atac, and U6atac, together with U5, are subunits of the so-called minor spliceosome that splices, only in metazoan (plant, insects and vertebrates), a rare class of pre-mRNA introns, denoted U12- type. Small nuclear RNA molecule can be (for example) complexed with a set of seven Sm or Sm-like proteins (Lsm), and several snRNP-specific proteins.

Spliceosomes are ribosome-sized (50 - 60 S) complexes composed of pre-mRNA, four small nuclear ribonucleoprotein (snRNP) particles, and a host of associated protein factors. The snRNPs (U1, U2, U4/6, and U5) are, in turn, multicomponent complexes, each containing at least one small stable RNA molecule (snRNA) and five or more tightly bound polypeptides. In all, it has been estimated that nuclear pre-mRNA splicing requires the action of over 100 different gene products

Having performed its task of exon ligation and mRNA production, the spliceosome disassembles before mRNA export. In addition, just as a machine is designed to perform repetitive tasks, the postcatalytic spliceosomal machinery must be reconfigured to allow a new round of splicing. The snRNP-bound lariat intron must be dissociated, allowing the lariat intron to be degraded and the snRNP to be recycled. Release of mRNA, disassembly, and recycling all necessarily involve extensive RNA:RNA rearrangements.

. 6. RNA interference (RNAi)

In order to target specific genes for silencing, siRNAs (small interfering, 21-23nt dsRNA) become the primary means. RNAi was first discovered in Caenorhabditis elegans, when it was noted that introducing a double-stranded RNA (dsRNA) that was homologous to a specific gene resulted in the post-transcriptional silencing of that gene. The obvious therapeutic potential of RNAi resulted in rapid elucidation of its mechanism of action: The dsRNA is initially recognised by an enzyme of the RNase III family of nucleases, named Dicer, and processed into small double-stranded molecules, termed siRNA and (ii) the siRNAs are bound by the RNA-induced silencing complex (RISC), which is a multi-protein complex (with RNase activity) that guides the targeted RNA to degradation. Although early investigations found that the introduction of long dsRNA had the potential to mediate the down-regulation of any gene, it was also found that in mammalian cells an anti-viral interferon response (IR) that resulted in the cessation of all protein synthesis was also elicited. Synthetic siRNAs (21–23 nucleotides in length) were shown not to elicit this IR and have hence been used in studies of mammalian gene function. More recently, plasmid and viral- based expression of small hairpin RNAs (shRNA) have been developed; these methods allow for longer-lived gene silencing effects and the use of viral vectors facilitates the use of RNAi in neuronal cells. Finally, comparing the activity of transfected siRNAs and virally delivered shRNAs shows that the efficacy of the RNAi effect is highly dependent on the cellular environment.

RNAi pathway:The first step in the RNAi pathway involves the processing of large dsRNAs into small, 21–23 nucleotide long siRNA molecules. Initial studies in Drosophila showed that an RNase III enzyme (known to recognise dsRNA) was responsible for this processing and that the siRNAs possessed 3´hydroxyl and 5´ phosphate groups and, importantly, a 3´ overhang of two unpaired nucleotides on each strand. A specific RNAase III enzyme was then found to be responsible for cleaving the dsRNAs and was named Dicer. The sequence homology and functional studies that followed led to the identification of Dicer homologues in Arabidopsis, Dictyostelium, fission yeast, C. elegans, mouse and human. A proposed model for the action of Dicer involves the ATP-dependent translocation of the enzyme along its dsRNA target. The efficiency with which Dicer cleaves a particular dsRNA molecule has also been shown to be directly proportional to the length of the target, since the longer the dsRNA, the greater the amount of siRNA produced and hence the more potent the silencing effect. This size limitation may prevent Dicer binding to small intramolecular base-paired regions of endogenous mRNAs. Human Dicer-mediated cleavage of dsRNA is thought to occur sequentially, beginning at the termini of the dsRNA, and by the excision of small dsRNA fragments of a defined length. Following the cleavage of dsRNA into siRNAs by Dicer the second important stage of mRNA degradation occurs. This is mediated by a protein complex with nuclease activity known as RISCwhich is guided to its target RNA by siRNA. This guide role of siRNA was proposed after the observation that dsRNA would only lead to the degradation of an mRNA with a homologous sequence, leaving the rest of the RNA in the cell unaffected. Moreover, it was shown that both siRNA and protein were required to mediate cleavage of the target. Following the initial discovery of the existence of a ribonucleoprotein complex as a mediator of RNAi, the components and mechanism of action of RISC began to be elucidated and both inactive and active forms of RISC complex (the active termed RISC*) were found. In addition a second ATP-dependent step was involved in the pathway and the following unwinding of the siRNA duplex, RISC was converted to RISC*. RISC* was found to be associated only with the antisense strand of the siRNA. Hence, although the siRNA needs to be double stranded in order to be efficiently recognised and bound to RISC, the two siRNA strands must unwind before RISC becomes active. Accordingly, it was concluded that either the RISC complex has ATP-dependent helicase activity or a helicase enzyme is associated with RISC. The efficient cleavage of the target mRNA by RISC was also shown to be dependent on the phosphorylation of the 5´ siRNA duplex. (In non-mammalian cells, there is evidence that an alternative branch of the RNAi pathway that results in the amplification of the original message can account for the efficiency of gene silencing. In this case, the unwound siRNA no longer acts as a guide to bring RISC to the target mRNA but merely as a primer for an RNA-dependent RNA polymerase (RdRP), which uses the target mRNA as a template to produce new dsRNA. This can subsequently be recognised and cleaved by Dicer, thus re-entering the RNAi pathway and initiating a new round of silencing. Therefore, not only is the mRNA targeted via the specific oligonuclotide sequence (and hence gene expression silenced) but also new dsRNAs arising from the entire mRNA sequence are created and thus amplify the original RNAi trigger. Several RdRPs participating in RNAi have been identified in fungi, plants and invertebrates However, evidence to suggest that a similar amplification mechanism is present in mammalian cells has not yet been found.)

Gene inhibition due to microRNAs (miRNA): miRNAs are short RNA molecules that prevent gene expression by inhibiting translation. The first two miRNAs to be studied were let-7 and lin-4, which control the expression of genes involved in developmental timing in C. elegans and Drosophila and hence were therefore also named small temporal RNAs. miRNAs are first transcribed as primary transccript or pri-miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem loop structures known as pre-miRNA in the . This processing is performed in mammalian cell by a protein complex known as the microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer. (This maturation step was shown to be catalysed by Dicer in a number of organisms, although with the aid of co-factors distinct from the RNAi pathway). The mature miRNA can then bind to the 3´-UTRs of their corresponding mRNAs, although their complementarity to the targets is not perfect as is the case for siRNA. A RISC-like complex was also shown to participate in the miRNA pathway. The RNAi machinery – and especially RISC – is also thought to be involved in mediating hetrochromatic and transposon silencing. Dicer and RISC can therefore be perceived as two central points, where several molecular pathways controlling gene expression converge.

7. Control of gene expression (control of transcription)

Prokaryotes

In bacteria, control of the rate of transcriptional initiation is the predominant site for control of gene expression. As with the majority of prokaryotic genes, initiation is controlled by two DNA sequence elements that are approximately 35 bases and 10 bases, respectively, upstream of the site of transcriptional initiation and as such are identified as the -35 and -10 positions. These 2 sequence elements are termed promoter sequences, because they promote recognition of transcriptional start sites by RNA polymerase. The consensus sequence for the -35 position is TTGACA, and for the -10 position, TATAAT. (The -10 position is also known as the Pribnow-box.) These promoter sequences are recognized and contacted by RNA polymerase.

The activity of RNA polymerase at a given promoter is in turn regulated by interaction with accessory proteins, which affect its ability to recognize start sites. These regulatory proteins can act both positively (activators) and negatively (repressors). The accessibility of promoter regions of prokaryotic DNA is in many cases regulated by the interaction of proteins with sequences termed operators. The operator region is adjacent to the promoter elements in most operons and in most cases the sequences of the operator bind a repressor protein. However, there are several operons in E. coli that contain overlapping sequence elements, one that binds a repressor and one that binds an activator.

Pprokaryotic genes that encode the proteins necessary to perform coordinated function are clustered into operons. Two major modes of transcriptional regulation function in bacteria (E. coli) to control the expression of operons. Both mechanisms involve repressor proteins. One mode of regulation is exerted upon operons that produce gene products necessary for the utilization of energy; these are catabolite-regulated operons. The other mode regulates operons that produce gene products necessary for the synthesis of small biomolecules such as amino acids. Expression from the latter class of operons is attenuated by sequences within the transcribed RNA.

Operon / theoperon model: As indicated above genes are often grouped into arrangements called operons. Operons consist of three parts: promoter (P) region (a region of RNA polymerase attachment); operator (O) site (a site of a repressor protein attachment; (If the repressor protein can attach to the operator, RNA polymerase cannot attach to the promoter and transcription of the genes in the third region of the operator can not happen.

The classic example of an operon is the so called lac operon. Some bacteria are able to use lactose as a source of energy. This requires a set of enzymes coded for by a series of genes (Z,Y,A) in the operon. The lac operon allows the transcription of these genes when lactose is present, and prevents transcription of these genes when lactose is absent. This works because of repressor protein (green) coded for by a so called regulatory gene (I). When lactose is absent, the regulatory protein has a conformation that allows it to fit into the operator site. This prevents the RNA polymerase from attaching to the promoter site. If lactose (an efector molecule) is present, a lactose molecule attaches to a binding site on the repressor protein. This alters the shape of the repressor protein (green/red mark) so that it cannot fit on the operator site. Thus, the RNA polymerase can attach to the promoter.

The z gene codes for β-galactosidase (β-gal), which is primarily responsible for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose and glucose. The y gene codes for permease, which increases permeability of the cell to β-galactosides. The a gene encodes a transacetylase.

(The lac operon is repressed, even in the presence of lactose, if glucose is also present. This repression is maintained until the glucose supply is exhausted. The repression of the lac operon under these conditions is termed catabolite repression and is a result of the low levels of cAMP that result from an adequate glucose supply. The repression of the lac operon is relieved in the presence of glucose if excess cAMP is added. As the level of glucose in the medium falls, the level of cAMP increases. Simultaneously there is an increase in inducer binding to the lac repressor. The net result is an increase in transcription from the operon. The ability of cAMP to activate expression from the lac operon results from an interaction of cAMP with a protein termed CRP (for cAMP receptor protein). The protein is also called CAP (for catabolite activator protein). The cAMP-CRP complex binds to a region of the lac operon just upstream of the region bound by RNA polymerase and that somewhat overlaps that of the repressor binding site of the operator region. The binding of the cAMP-CRP complex to the lac operon stimulates RNA polymerase activity 20-to-50-fold).

The trp operon: encodes the genes for the synthesis of tryptophan. This cluster of genes, like the lac operon, is regulated by a repressor that binds to the operator sequences. The activity of the trp repressor for binding the operator region is enhanced when it binds tryptophan; in this capacity, tryptophan is known as a corepressor. Since the activity of the trp repressor is enhanced in the presence of tryptophan, the rate of expression of the trp operon is graded in response to the level of tryptophan in the cell.

Attenuation: The attenuator region, which is composed of sequences found within the transcribed RNA, is involved in controlling transcription from the operon after RNA polymerase has initiated synthesis. The attenuator of sequences of the RNA are found near the 5' end of the RNA termed the leader region of the RNA. The leader sequences are located prior to the start of the coding region for the first gene of the operon (the trpE gene). The attenuator region contains codons for a small leader polypeptide, that contains tandem tryptophan codons. This region of the RNA is also capable of forming several different stable stem-loop structures. Depending on the level of tryptophan in the cell and hence the level of charged trp-tRNAs, the position of on the leader polypeptide and the rate at which they are translating allows different stem-loops to form. If tryptophan is abundant, the ribosome prevents stem-loop 1-2 from forming and thereby favors stem-loop 3-4. The latter is found near a region rich in uracil and acts as the transcriptional terminator loop. Consequently, RNA polymerase is dislodged from the template.

The operons coding for genes necessary for the synthesis of a number of other amino acids are also regulated by this attenuation mechanism. It should be clear, however, that this type of transcriptional regulation is not feasible for eukaryotic cells.

The ara operon: The structural genes (araB, araA, and araD) that encode the metabolic enzymes that break down arabinose are transcribed as a multigenic mRNA. Transcription is activated at araI, the initiator region, which contains both an operator site and a promoter. The araC gene encodes an activator protein that, when bound to arabinose, activates transcription of the ara operon, perhaps by helping RNA polymerase bind to the promoter, located within in the araI region. An additional activation event is mediated by the same CAP cAMP catabolite repression system that regulates lac operon expression. (In the presence of arabinose, both the CAP cAMP complex and the AraC arabinose complex must bind to the initiator region in order for RNA polymerase to bind to the promoter and transcribe the ara operon).

In the absence of arabinose, the AraC protein assumes a different conformation and represses the ara operon by binding both to araI and to a second operator region, araO, thereby forming a loop that prevents transcription. Thus, the AraC protein has two conformations, one that acts as an activator and the other that acts as a repressor. The two conformations, dependent on whether the allosteric effector has bound to the protein, also differ in their abilities to bind a specific target site in the araO region of the operon.

Eukaryotes

The presence of a nuclear membrane prevents the simultaneous transcription and translation that occurs in prokaryotes. Whereas, in prokaryotes, control of transcriptional initiation is the major point of regulation, in eukaryotic cells, the ability to express biologically active proteins comes under regulation at several points:

1. Chromatin structure: The physical structure of the DNA, as it exists compacted into chromatin, can affect the ability of transcriptional regulatory proteins (termed transcription factors) and RNA polymerases to find access to specific genes and to activate transcription from them. The presence modifications of the histones and of CpG methylation most affect accessibility of the chromatin to RNA polymerases and transcription factors.

2. Epigenetic control: Epigenesis refers to changes in the pattern of gene expression that are not due to changes in the nucleotide composition of the genome. Literally "epi" means "on" thus, epigenetics means "on" the gene as opposed to "by" the gene.

3. Transcriptional initiation: This is the most important mode for control of eukaryotic gene expression (see below for more details). Specific factors that exert control include the strength of promoter elements within the DNA sequences of a given gene, the presence or absence of enhancer sequences (which enhance the activity of RNA polymerase at a given promoter by binding specific transcription factors), and the interaction between multiple activator proteins and inhibitor proteins.

4. Transcriptp processing and modification: Eukaryotic mRNAs must be capped and polyadenylated, and the introns must be accurately removed. Several genes have been identified that undergo tissue-specific patterns of alternative splicing, which generate biologically different proteins from the same gene.

5. RNA transport: A fully processed mRNA must leave the nucleus in order to be translated into protein.

6. Transcript stability: Unlike prokaryotic mRNAs, whose half-lives are all in the range of 1 to 5 minutes, eukaryotic mRNAs can vary greatly in their stability. Certain unstable transcripts have sequences (predominately, but not exclusively, in the 3'-non- translated regions) that are signals for rapid degradation.

7. Translation initiation: Since many mRNAs have multiple methionine codons, the ability of ribosomes to recognize and initiate synthesis from the correct AUG codon can affect the expression of a gene product. Several examples have emerged demonstrating that some eukaryotic proteins initiate at non-AUG codons. This phenomenon has been known to occur in E. coli for quite some time, but only recently has it been observed in eukaryotic mRNAs.

8. Smal RNAs and control of transcript levels: Within the past several years a new model of gene regulation has emerged that involves control exerted by small non- coding RNAs. This small RNA-mediated control can be exerted either at the level of the translatability of the mRNA, the stability of the mRNA or via changes in chromatin structure.

9. Post-translational modification: Common modifications include glycosylation, acetylation, fatty acylation, disulfide bond formations, etc.

10. Protein transport: In order for proteins to be biologically active following translation and processing, they must be transported to their site of action.

11. Control of protein stability: Many proteins are rapidly degraded, whereas others are highly stable. Specific amino acid sequences in some proteins have been shown to bring about rapid degradation.

(see also Chapters 5. and 6.)

Epigenetic control of gene expression: the term epigenetics is used to define the mechanism by which changes in the pattern of inherited gene expression occur in the absence of alterations or changes in the nucleotide composition of a given gene. A literal interpretation is that epigenetics mean "in addition to changes in genome sequence." The easiest way to understand this concept is to think about the fertilized egg: at the moment of fertilization that single cell is totipotent, i.e. as it divides the daughter cells ultimately differentiate into all the different cells of the organism. The only difference between the various cells of the resultant organism are the consequences of differential gene expression, not due to differences in the sequences of the genes themselves. Evidence indicates that most of the epigenetic modifications are erased during gametogenesis and/or following fertilization. Several different types of epigenetic events have been identified. As described in the section above relating to chromatin structure as a means to control gene expression and the role of DNA methylation in these structural changes, DNA methylation is likely to be the most important epigenetic event controlling and importantly maintaining the pattern of gene expression during development. Other DNA modification events are also known to effect epigenetic phenomena including acetylation, methylation phosphorylation, ubiquitylation and sumoylation of histone proteins. Thus, it should be clear that the same events that affect chromatin structure can be defined as epigenetic events. An additional process that affects chromatin structure and therefore gene expression is considered an epigenetic event and this involves the small interfering RNAs (siRNAs)

.

8. Conjugative gene transfer

Conjugation (bacterial) is the transfer of genetic material between bacteria through direct cell-to-cell contact. Discovered in 1946 by Joshua Lederberg and Edward Tatum, conjugation is a mechanism of horizontal gene transfer (HGT) - as are transformation and transduction - although these mechanisms do not involve cell-to-cell contact. Bacterial conjugation is often incorrectly regarded as the bacterial equivalent of sexual reproduction or mating. It is not actually sexual, as it does not involve the fusing of gametes and the creation of a zygote, nor is there equal exchange of genetic material. It is merely the transfer of genetic information from a donor cell to a recipient. In order to perform conjugation, one of the bacteria, the donor, must play host to a conjugative or mobilizable genetic element, most often a conjugative or mobilizable plasmid or transposon. Most conjugative plasmids have systems ensuring that the recipient cell does not already contain a similar element.The genetic information transferred is often beneficial to the recipient cell. Benefits may include resistance, other xenobiotic tolerance, or the ability to utilize a new metabolite. Such beneficial plasmids may be considered bacterial endosymbionts. Someconjugative elements may also be viewed as genetic parasites on the bacterium, and conjugation as a mechanism that was evolved by the mobile element to spread itself into new hosts.

The prototype for conjugative plasmids is the F-plasmid, also called the F-factor. The F-plasmid is an episome (a plasmid that can integrate itself into the bacterial by genetic recombination) of about 100 kb length. It carries its own origin of replication, the oriV, as well as an origin of transfer, or oriT. There can only be one copy of the F-plasmid in a given bacterium, either free or integrated (two immediately before cell division). The host bacterium is called F-positive or F-plus (denoted F+). Strains that lack F plasmids are called F-negative or F-minus (F-). Among other genetic information, the F-plasmid carries a tra and a trb locus, which together are about 33 kb long and consist of about 40 genes. The tra locus includes the pilin gene and regulatory genes, which together form pilion the cell surface, polymeric proteins that can attach themselves to the surface of F- bacteria and initiate the conjugation. Though there is some debate on the exact mechanism, the pili themselves do not seem to be the structures through which the actual exchange of DNA takes place; rather, some proteins coded in the tra or trb loci seem to open a channel between the bacteria. When conjugation is initiated, via a mating signal, a relaxase enzyme creates a nick in one plasmid DNA strand at the origin of transfer, or oriT. The relaxase may work alone or in a complex of over a dozen proteins, known collectively as a relaxosome. In the F-plasmid system, the relaxase enzyme is called TraI and the relaxosome consists of TraI, TraY, TraM, and the integrated host factor, IHF. The transferred, or T-strand, is unwound from the duplex plasmid and transferred into the recipient bacterium in a 5'-terminus to 3'-terminus direction. The remaining strand is replicated, either independent of conjugative action (vegetative replication, beginning at the oriV) or in concert with conjugation (conjugative replication similar to the rolling circle replication of lambda phage). Conjugative replication may necessitate a second nick before successful transfer can occur. A recent report claims to have inhibited conjugation with chemicals that mimic an intermediate step of this second nicking event. If the F-plasmid becomes integrated into the host genome, donor chromosomal DNA may be transferred along with plasmid DNA. The certain amount of chromosomal DNA that is transferred depends on how long the bacteria remain in contact; for common laboratory strains of E.coli the transfer of the entire bacterial chromosome takes about 100 minutes. The transferred DNA can be integrated into the recipient genome via recombination A culture of cells containing non-integrated F plasmids usually contains a few that have accidentally become integrated, and these are responsible for those low-frequency chromosomal gene transfers which do occur in such cultures. Some strains of bacteria with an integrated F-plasmid can be isolated and grown in pure culture. Because such strains transfer chromosomal genes very efficiently, they are called Hfr (high frequency of recombination. The E.coli genome was originally mapped by interrupted mating experiments, in which various Hfr cells in the process of conjugation were sheared from recipients and investigating which genes were tranferred.

F-type sex pili: The pili encoded by F plasmid in are involved in formation of cell aggregates as a prelude to conjugative gene transfer from the F-plasmid containing cells to a recipient (see figure). The F-pilus itself has an elaborate structure comprising several different proteins, encoded by numerous genes on the F-plasmid that are involved in pilus formation and DNA transfer, including genes traA, E, K, B, V, W, U, F and G.Adhesion of bacterial cells has an important survival role in their survival as micro-colonies - called biofilms - on solid surfaces in the natural environment, and F-pili determine the final shapes of the structures seen in mature surface biofilms formed by Eschericha coli bacteria, as mutants affected in the plasmid specified F-pili form a biofilm of a different structure. The F- pilus allows for the transfer of a single strand of bacterial DNA from the F-piliated (donor) bacteria to the recipient bacteria by conjugation where it is converted to the double-stranded version of DNA. Similar gene transfer abilities are carried by many different plasmids such as the R-plasmids that confer antibiotic resistance. Through this mechanism of conjugation based gene transfer, advantageous genetic traits can be widely disseminated amongst populations of bacteria.

sex pili

surface structures

cell – cell contact

DNA transfer

control

Conjugative transposons are integrated DNA elements that excise themselves to form a covalently closed circular intermediate. This circular intermediate can either reintegrate in the same cell (intracellular transposition) or transfer by conjugation to a recipient and integrate into the recipient's genome (intercellular transposition). They are now known to be present in a variety of gram- positive and gram-negative bacteria also, they have a surprisingly broad host range, and they probably contribute as much as plasmids to the spread of antibiotic resistance genes in some genera of bacterial pathogens.disease-causing bacteria. Many conjugative transposons can mobilize coresident plasmids. Conjugative transposons are transposon-like in the sense that they excise from and integrate into DNA, but they appear to have a different method of excision and integration from that of well-studied transposons such as Tn5 and Tn10, in their mechanism of excision and integration. For example, conjugative transposons have a covalently closed circular transposition intermediate and do not duplicate the target site when they integrate into DNA. Conjugative transposons are plasmid-like in that they have a covalently closed circular transfer intermediate and are transferred by conjugation, but unlike plasmids, the circular intermediate of a conjugative transposon does not replicate, at least in hosts so far investigated. This caveat is an important one, because there are plasmids in Streptomyces spp. that integrate into the chromosome in some hosts and replicate as plasmids in other hosts. At this point, the possibility that the circular forms of conjugative transposons are capable of replication in some hosts cannot be ruled out. In fact, conjugation itself could be viewed as a form of replication, because the single-stranded circle that remains behind in the donor and the single-stranded copy that enters the recipient must be made double stranded before integration occurs. Conjugative transposons are phage-like in that their excision and integration resembles excision and integration of temperate bac phages, which also have a circular intermediate. In fact, sequence analysis of integrases of some conjugative transposons suggests that they are members of the lambda integrase family. In contrast to the lambdoid phages, however, conjugative transposons do not form viral particles, and they are transferred by conjugation rather than by phage transduction. The purpose of this review is to survey recent work on the characteristics and activities of conjugative transposons.

9. DNA damage response

DNA damage: both metabolic activity and environmental factors can cause DNA damage, i.e., structural damage to the DNA molecule affecting its function. In this connection, DNA repair refers to a collection of processes by which a cell identifies and corrects such lesions. Therefore, the DNA repair ability of a cell is vital to the integrity of its genome and thus to its normal functioning. When repair processes fail, and when cellular apoptosis does not take effect, irreparable DNA damages can occur, including double-strand breaks, DNA crosslinkages, among others. Their accumulation can induce: apotosis (see Chapter 4.), unregulated cell division, irreversible state of dormancy… DNA damage mostly affects the primary structure of the double helix; that is, the bases themselves are chemically modified. These modifications can in turn disrupt the molecules' regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. These damages and distubances consequently affect DNA superstructures (DNA is supercoiled).

DNA damage due to endogenous cellular processes: alkylation of bases (usually methylation), such as formation of 7-methylguanine, 1-methyladenine, 6-O-methylguanine; oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] and generation of DNA strand interruptions from reactive oxygen species; hydrolysis of bases, such as deamination, depurination and depyrimidination; "bulky adduct formation" (i.e. benzo[a]pyrene diol epoxide-dG adduct); mismatch of bases, due to errors in DNA replication, in which the wrong DNA base is stitched into place in a newly forming DNA strand, or a DNA base is skipped over or mistakenly inserted.

DNA damage caused by exogenous agents: UV-A-light creates mostly free radicals. The damage caused by free radicals is called (indirect DNA damage); UV-B-light causes crosslinking between adjacent cytosine and thymine bases creating pyrimidine dimers (direct DNA damage); Ionizing radiation such as that created by radioactive decay causes breaks in DNA strands; Thermal disruption at elevated temperature increases the rate of depurination (loss of purine bases from the DNA backbone) and single strand breaks. Xenobiotics / create a huge diversity of DNA adducts- ethenobases, oxidized bases, alkylated phosphotriesters and crosslinking of DNA, just to name a few.

DNA repair

Direct reversal mechanisms: are specific to the type of damage that does not involve breakage of the phosphodiester backbone. These mechanisms do not require a template, since the types of damage they counteract can only occur in one of the four bases. The formation of thymine dimers (a common type of cyclobutyl dimer) upon irradiation with UV light results in an abnormal covalent bond between adjacent thymidine bases (above). The photoreactivation process directly reverses this damage by the action of the enzyme photolyase, whose activation is obligately dependent on energy absorbed from blue /UV light (300–500 nm) to promote catalysis. Another type of damage, methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase. This is an expensive process because each enzyme molecule can only be used once; that is, the reaction is stoichiometric rather than catalytic A generalized response to methylating agents in bacteria is known as the adaptive response and confers a level of resistance to alkylating agents upon sustained exposure by upregulation of alkylation repair enzymes. The third type of DNA damage reversed by cells is certain methylation of the bases cytosine and adenine.

Single strand damage: if one of the two strands of a double helix is damaged, the other strand can be used as a template to guide the correction of the damaged strand. In order to repair damage to one of the two paired molecules of DNA, there exist of excision repair mechanisms that remove the damaged nucleotide and replace it with an undamaged nucleotide complementary to that found in the (undamaged) templte DNA strand.

(BER) Base excision repair: repairs damage to a single base caused by oxidation, alkylation, hydrolysis, or deamination. The damaged base is removed by a DNA glycolyase, resynthesized by a DNA polymerase, and a DNA ligase performs the final nick-sealing step.

(NER) Nucleotide excision repair: which recognizes bulky, helix-distorting lesions such as pyrimidine dimers and other photoproducts. A specialized form of NER known as transcriptin –coupled repair deploys NER enzymes to genes that are being actively transcribed.

(MMR) Mismatch repair: which corrects errors of DNA replication and recombination that result in mispaired (but undamaged nucleotides.

BER

NER

MMR

10. Cell – Cell signaling

Cell signaling: The ability of cells to perceive and respond to external stimuli. (Cells respond to stimuli via cell signaling). The overall flow of information during cell signaling: Binding of ligand by a receptor activates a series of events known as signal transduction, which relays the signal to the interior of the cell, resulting in specific cellular responses and / or changes in gene expression. Signal transduction pathways consist of a series of steps: recognition of the stimulus by a specific plasma membrane receptor.Transfer of a signal across the plasma membrane (casacde of membrane proteins conformational changes).Transmission of the signal to effector molecules within the cell, which causes a change in cellular activities. Cessation of the cellular response due to inactivation of the signal molecule. Each cell is programmed to respond to specific combinations of exreaceluular signal molecules. Different cells can respond differently to the same extracellular signal molecule Different types of chemical signals can be received by one receptor / different receptors. Second messengers (cAMP), an effector, amplify the response to a single extracellular ligand by cAMP to trigger a reaction cascade.The cascade starts with the binding of cAMP to cAMP-dependent protein kinase

cell-to-cell communication: requires direct cell – cell contacts or cells communicate with each other over short distances (paracrine signaling), or over large distances (endocrine siganaling);).Some cells can form gap junction that connect their cytoplasm to the cytoplasm of adjacent cells. Another form is the autocrine signaling in which a cell secretes a signal molecule (called the autocrine agent) that binds to autocrine receptors on the same cell type as the emitting cell.

G protein-linked receptors: are exposed at the extracellular surface of the cell membrane and contain seven transmembrane segments that form a ligand-binding site on the outside of the cell and a G protein-binding site on the inside. In the resting (unstimulated) state, the cytoplasmic domain of the receptor is noncovalently linked to a G protein that consists of α and βγ subunits. Upon activation, the α subunit exchanges GDP for GTP. The α-GTP subunit then dissociates from the βγ subunit, and the α or βγ subunit diffuses along the inner leaflet of the plasma membrane to interact with a number of different effectors.These effectors include adenylyl cyclase, phospholipase C, various ion channels, and other classes of proteins. Signals mediated by G proteins are usually terminated by the hydrolysis of GTP to GDP, which is catalyzed by the inherent GTPase activity of the α subunit . One major role of the G proteins is to activate the production of second messengers, that is, signaling molecules that convey the input provided by the first messenger (ligand). The activation of cyclases, such as adenylyl cyclase, which catalyzes the production of the second messenger cyclic adenosine- 3′,5′-monophosphate (cAMP), and guanylyl cyclase, which catalyzes the production of cyclic guanosine-3′,5′-monophosphate (cGMP), constitutes the most common pathway linked to G proteins. (In addition, G proteins can activate the enzyme phospholipase C (PLC) which, among other functions, plays a key role in regulating the concentration of intracellular calcium. Upon activation by a G protein, PLC cleaves the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) to the second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). IP3 triggers the release of Ca2+ from intracellular stores, thereby dramatically increasing the cytosolic Ca2+ concentration and activating downstream molecular and cellular events. DAG activates protein kinase C, which then mediates other molecular and cellular events, including smooth muscle contraction and transmembrane ion transport. All of these events are dynamically regulated, so that the different steps in the pathways are activated and inactivated with characteristic kinetics.) A large number of Gα protein isoforms have now been identified, each of which has unique effects on its targets. A few of these G proteins include G-stimulatory (Gs), G-inhibitory (Gi), Gq, Go, and G12/13. The differential functioning of these G proteins, some of which may couple in different ways to the same receptor in different cell types, is likely to be important for the potential selectivity of future drugs. The βγ subunits of G proteins can also act as second messenger molecules, although their actions are not as thoroughly characterized.

Alternatively:

Receptor tyrosine kinases: (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligand.. (RTK proteins are classified into subfamilies depending on their structural properties and ligand specificity.) RTKs need to form dimers (stabilized by ligand binding by the receptor) in the plasma membrane. Interaction between the two cytoplasmic domains of the dimer is thought to stimulate autophosphorylation of tyrosines within the cytoplasmic tyrosine kinase domains of the RTKs causing their conformational changes. The kinase domain of the receptors is subsequently activated, initiating signaling cascades of phosphorylation of downstream cytoplasmic molecules. (The mutation of certain RTK genes can result in the expression of receptors that exist in a constitutively-activate state. Such mutated RTK genes may act as oncogens, genes that contribute to the initiation or progression of cancer.)

A ligand-activated ion channels: recognizing its ligand undergo a conformational / structural change that opens the plasma membrane channel through which ions can pass. In addition, calcium ions are also commonly allowed into the cell during ligand-induced ion channel opening. This calcium can act as a classical second messenger, setting in motion signal transduction cascades and altering the cellular physiology of the responding cell.

11. Bacterial chemotaxis

Generally, the process by which bacterial cells sense chemical gradients in their environment and then move towards more favorable conditions. In chemotaxis, events at the receptors control autophosphorylation of the CheA histidine kinase, and the phosphohistidine is the substrate for the response regulator CheY, which catalyzes the transfer of the phosphoryl group to a conserved aspartate. The resulting CheY-P can interact with the switch mechanism in the motor. This interaction causes a change in behavior, such as in direction or speed of rotation of flagella. In bacteria and , motility is controlled by a two-component system involving ahistidine kinase that senses the environment and a response regulator, a very common type of signal transduction in prokaryotes. Most insights into the processes involved have come from studies of Escherichia coli over the last three decades. However, in the last 10 years, with the sequencing of many prokaryotic genomes, it has become clear that E. coli represents a streamlined example of bacterial chemotaxis. While general features of excitation remain conserved among bacteria and archaea, specific features, such as adaptational processes and hydrolysis of the intracellular signal CheY-P, are quite diverse. The Bacillus subtilis chemotaxis system is considerably more complex and appears to be similar to the one that existed when the bacteria and archaea separated during evolution, so that understanding this mechanism should provide insight into the variety of mechanisms used today by the broad sweep of chemotactic bacteria and archaea. However, processes even beyond those used in E. coli and B.subtilis have been discovered in other organisms.

(Bacteriaal cell) flagellar rotation: alternates between the default direction of counter- clockwise (CCW) and clockwise (CW) rotation. . The process by which bacteria control the frequency of switching between CCW and CW flagellar rotation in response to chemical gradients is called chemotactic signalling. In a chemically homogeneous environment, the cells change direction approximately once per second, and there is no bias for net movement in any particular direction. However, in the presence of a concentration gradient of chemoattractant or chemorepellent, this frequency is altered, enabling bacteria to swim up concentration gradients of attractants and down concentration gradients of repellents. Net movement is achieved by lengthening the period of runs as a cell is experiencing an increasing concentration of attractant, and decreasing the period of runs when there is a decreasing concentration of attractant (below-b, c)

Chemotaxis receptors (Escherichia coli model): are called methyl-accepting chemotaxis proteins (MCPs). These are generally transmembrane proteins, although cytoplasmic MCPs have also been found,. An important feature of MCPs is their clustering mostly at the cell poles. MCPs bind specific ligands and ligand occupancy is communicated to the flagella through a signal-transduction cascade (below). Bacteria can respond to changes of only a few molecules of ligand and clustering is thought to be important not only for this sensitivity but also for the signal amplification that is required to achieve efficient chemotaxis. A decrease in attractant binding to MCPs is communicated to the auto-histidine kinase CheA through the protein CheW, resulting in auto-phosphorylation of CheA to CheA-P. CheA and CheW form a ternary complex with the MCPs at the cell poles. Two soluble response regulators, CheY and CheB, compete for phosphorylation by CheA-P. As CheY-P binds to the flagellar switch protein FliM (see Chapter 12) and causes CW rotation, the intracellular ratio of CheY to CheY-P controls the direction of flagellar rotation. Tumbles are kept brief through rapid CheZ-stimulated dephosphorylation of CheY-P, resulting in turnover of this CW-promoting signal. The adaptation to further increases or decreases in attractant binding (crucial for chemotaxis along gradients) is achieved in E. coli by modulating the methylation state of the MCPs using two proteins, a constitutively active methyltransferase CheR and a methylesterase CheB, the activity of which is stimulated after phosphorylation by CheA-P. Increased methylation of the MCPs dampens the response to ligand binding, whereas decreased methylation sensitizes this response. This allows for adaptation because the decision-making reaction (CheA auto-phosphorylation) can be set to approximately the pre- stimulus level.

12. Bacterial cell motility

By far the most common means of locomotion in microorganisms is the flagella, that allow microorganisms to propel themselves through the expense of their chemiosmotic gradient. Additionally there is the mysterious movement through a process known as ―gliding‖, and vertical motion through control of gas . Gliding: not well understood form of motion, seen as a means of moving along surfaces, not though a liquid medium (Cyanobacteria are some of the most well known gliders). This motion is effected by the secretion of a polysaccharide solution that binds to the solid surface. As the secreted goo, which binds to both the cell and to the surface the cell is gliding along, binds to the solid substrate the cell tends to get pulled in one direction or another. By controlling where the goo is secreted, the cell can control in which direction it moves.According to second model gliding involves the motion of large protein complexes that are attached through the different layers of cell. These proteins bind to the substrate, and then are driven to the ―back‖ of the cell. microorganisms in the ocean undergo a daily migration to lower depths in the day and a return in the evening to avoid predators. Gas vesicle motility: Cell ppopulations of marine taxons undergo a regular migration to lower depths in the day and a return in the evening to avoid predators. By controlling their density, the cell of these populations can control their position in the water column. Seawater becomes more dense the deeper it gets, both by compression and by varying concentrations of solutes. Unicellular organisms use this to their advantage by adjusting their net density. Gas filled vesicles are in many unicellular organisms, and adjustment of the size of these vesicles will adjust the net density of the organism.

(Bacterilal) flagelar motility:

Three different forms of flagella have been described, corresponding to the three branches of the tree of life. Eukaryotic flagella are whip like, and propel their cells in explosive movements. In contrast, Bacterial flagella are comparatively stiff helical structures that rotate, generating propulsion. Archaeal flagella are superficially similar to Bacterial flagella, however have numerous differences. There are several different gross morphologies of Bacterial flagella, based upon the number and placement of individual flagella. While the actual mechanism of each flagella unit is fairly common across species, the placement and structure varies, making it a common feature by which to classify bacterial species. Some bacterial cells only have one flagella. These species are considered monotrichous, while other cells have two flagella on opposite sides of a cell and are considered amphitrichous. Both of these arrangements are also considered to be polar arrangements. In addition, flagella are present in a single clump, an arrangement that is termed lophotrichous (see Chapter 11.). These units often wrap together to make what is effectively a single larger screw drive. Peritrichous flagella are arranged all over the cell. The other major means of differentiation comes within the characterization of the helix that is formed by the filament section of the flagella unit. These filaments will have a characteristic undulation that described as a wavelength. This wavelength can be used to determine the species of bacteria that is being examined. However, while there are differences between flagella in these respects, the basic structure and mechanism of flagella function remains similar. The flagella consists of three main parts: The long helical filament which provides the interface transform rotational motion into linear motion; the hook, which connects the filament to the motor complex; and the motor complex, which provides the rotational motion. Of these three, the motor complex shows the most variation. Since it is a transmembrane protein, it needs to transverse all layers which surround the cell, thus it is significantly different in gram-negative proteins and gram-positive proteins.

In gram positive bacteria, there are three structures that allow a central rod to bypass the two layers that separate the extracellular environment from the cytoplasm. The central rod acts like an axle to transfer the rotation to the outside of the cell. In the cytoplasmic membrane, the two structures are the MS ring and the C ring. The C ring is located in the cytoplasm, and is connected to the MS ring via the central rod. It is on the cytoplasmic layer that the turning force is generated. In Bacteria, it is generated via the discharging of the chemiosmotic gradient. A concentration of protons in the periplasmic or extracellular are allowed to flow back in by forcing the rotor to turn. The protons are thought to be brought back in via the Mot proteins, while the Fli proteins act as on and off switches. In Archaea, the same basic system is used, except the rotary motion is not generated by the chemiosmotic gradient, but instead by the hydrolysis of ATP.

The P ring is the structure that allows the rotary motion to be translated through the layer. It is embedded in the peptidoglyan and does not rotate along with the C ring and the MS ring. In Gram Negative bacteria, there is yet another layer to bypass, so another cuff is needed, this one is called the L ring. Thus the P and L rings are essentially low friction cuffs that allow the rod to rotate; the C ring, MS ring and the rod are the rotor and the Mot proteins are the stator. Through the center of the whole complex runs a hollow rod. It is through this rod that flagellin proteins travel so that they can travel to the point of flagella synthesis. In Bacteria flagella are synthesized at the tip, not at the base. However, in Archaea flagella grow from the base. Furthermore, the size of the filament in Archaea is much smaller than in Bacteria, and is in fact too small to allow flagellin to flow through the tube. Movement by flagella is very fast. Bacteria powered by flagella are able to propel themselves at speeds of 60 cell lengths a second. Furthermore, some flagella are capable of rotating in both directions, and thus creating movement in both directions. The swimming pattern of bacteria, such as Escherichia coli and Salmonella, consists of straight swimming for a few seconds and tumbling for a fraction of a second. During the straight swimming phase, the helical filaments form a bundle behind the cell body, where the filaments are all in a left- handed supercoiled form, each acting as a propeller driven by a rotary motor at the base of the . Bacteria tumble once every few seconds to change their swimming direction for the chemotactic behavior. The tumbling is triggered by quick reversal of motor rotation, which produces a twist in the filament structure and transforms it into right-handed supercoils momentarily. This allows the bundle to fall apart smoothly and then the uncoordinated propelling forces change the orientation of the cell quickly. The flagellar filaments can also be transformed into various but distinct polymorphic forms including two straight forms, in response to amino acid replacements in flagellin5 and to chemical changes in the environment.

The flagellum is built from the inside out. First, there is activated transcription of the genes that encode the components of the inner . A checkpoint mechanism monitors assembly of the early flagellum structure, which results in activation of the response regulator by phosphorylation when the inner basal body is assembled. Activated reguator activates the transcription of genes that encode the outer basal body and the hook. Finally, the genes that encode the filament proteins. These genes are expressed only if the basal body and the hook is assembled.

Both the hook and the helical filament are self-assembling macromolecular structures composed of the hook protein (FlgE) and flagellin (FliC), respectively. Each filament may comprise as many as ~30000 flagellin subunits and can grow up to ~15 μm. The hook is a helical assembly of ~130 copies of FlgE subunits with a well regulated length of 55 nm ± 6 nm, capable of forming polymorphic supercoil structures. Bacterial flagellar hook acts as a molecular universal joint that can transmit the torque produced by the basal body, a rotary motor, to the flagellar filament. Other components of the filamentous axial portion of the bacterial flagellum are the five rod proteins (FliE, FlgB, FlgC, FlgF, FlgG) and the hook associated proteins HAP1 (FlgK), HAP2 (FliD) and HAP3 (FlgL). HAP1 and HAP3 are junction proteins that connect the hook to the filament. HAP2 forms a capping structure at the distal end of the flagellar filament, which helps to incorporate flagellin monomers—transported through the central channel—into the filament at the tip.

The flagellar proteins forming the structures lying beyond the cytoplasmic membrane are synthesized in the cell and exported sequentially by the flagellum-specific protein export apparatus from the cytoplasm to the site of assembly at the distal end of the growing filament. They must be translocated through the narrow (20-25 Å wide) central channel of the flagellum in mostly unfolded conformation. The flagellar protein export system is thought to exist at the cytoplasmatic face of the basal body to distinguish flagellar proteins from other cytoplasmatic proteins and to facilitate their transportation.

The assembly process of the bacterial flagellum starts from the formation of the FliF ring complex (also called the MS ring) of the basal body in the cytoplasmic membrane and proceeds in both inward and outward directions, as well as laterally. The inward assembly involves the formation of the C ring, which is also called the switch complex, in the cytoplasmic space and the flagellar export apparatus is formed within the C ring, getting ready to export flagellar axial proteins through its central channel.

For all axial structure formation, the flagellum-specific type III protein export system selectively binds and translocates flagellar axial proteins into the central channel of the flagellum. Since the identification and enzymatic characterization of FliI ATPase as a component of the flagellar protein export system, it had been thought that the flagellar protein export is driven by the energy of ATP hydrolysis. A recent study, however, clearly showed that the flagellar proteins are exported to form the flagellum even in the absence of FliI and that the proton motive force across the cytoplasmic membrane is responsible for driving most part of the export process that involves unfolding of export substrate proteins and translocation of the unfolded chains, with a help of the FliI hexamer ring complex for insertion of the NH2-terminal chain of the export substrate proteins. The axial proteins construct the rod, the hook, the hook-filament junction and the long filament in this order. The rod composed of five proteins is connected to the FliF ring at its proximal portion and with the hook at its distal end and traverses the periplasmic space through the LP ring, where the P ring is located within the peptidoglycan (PG) layer and the L ring within the outer membrane. The rod cap made of FlgJ not only facilitates rod protein assembly but also makes a local hole through the PG layer by its muramidase activity, thus permitting penetration of the rod through the PG layer. Then FlgE assembles in a helical manner to form the hook. The hook cap made of FlgD is attached at the distal end of the hook until the hook grows up to a length of about 55 nm. The FliK and FlhB proteins function together to control the hook length within 10% of the average length. The hook length is determined at the point that the export system switches its export specificity from recognition of early substrates called rod/hook type (rod proteins, FlgJ, hook protein, FlgD, FliK) to late substrates called filament type (FlgM, HAP proteins, flagellin). Then the FlgD cap falls off the tip of the hook and is replaced by HAP1 and then HAP3 and HAP2 are bound at the distal end in this order to form the basal body-hook-HAP1-HAP3-HAP2 complex momentarily before the initiation of flagellin assembly into the long helical filament. (The flagellar filament is composed of a single protein, flagellin. Flagellin from a wild-type strain of Salmonella, SJW1103, is composed of 494 amino acids. The amino acid sequences of the terminal regions of flagellin, including about 180 NH2-terminal and 100 COOH-terminal residues, are known to be well conserved from species to species of bacteria, while the central region is highly variable.)

Flagellin monomers travel a long way through the narrow central channel, only 2 nm in diameter but up to 10-15 μm long, to the flagellar tip, presumably by a diffusion process. Filament assembly in vivo requires the presence of the HAP2 pentamer complex capping the distal end of the filaments where flagellin monomers are assembled. The cap permits assembly of flagellin and prevents its excretion. The simplest role of the cap is to prevent subunits from diffusing away from the tip but it may also induce a conformational change in flagellin required for polymerization. Interactions between the cap and distal end of the filament seem to have unique characteristics. The binding is supposed to be very stable so that dissociation of the cap rarely occurs and yet flagellin monomers can be easily inserted between the HAP2 cap and the end of the filament.

Flagellar axial proteins can be divided into three major families. Hook protein shows sequential similarities, especially in its terminal regions, to the rod proteins (FliE, FlgB, FlgC, FlgF, FlgG), which compose the drive shaft of the flagellar motor and also to the proximal hook associated protein, HAP1. Flagellin is not similar to the hook family, but appears to be related to HAP3. HAP2 is the most dissimilar among the axial proteins; it shows no significant sequential homology to flagellin or hook protein.

The flagelar filament is composed of 11 strands of protofilaments, which are nearly longitudinal arrays of subunits. The densely packed central core of filament consists of a concentric double tubular structure made of domains D0 and D1. Domains D2 and D3, which project out from the filament core, are relatively well separated from one another. The diameter of the filament is approximately 230 Å and that of the central channel is about 20 Å.

The bacterial flagellar hook acts as a molecular universal joint, transmitting torque produced by the rotary motor to the filament. The hook is much more flexible than the filament. The hook, like the helical filament, is also an assembly of 11 circularly arranged protofilaments and shows polymorphic supercoiling, which is only visible in polyhook structures. A specific aspect of the hook structure is its very small radius of curvature and short helical pitch. The most plausible mechanical explanation of how the hook works as a universal joint is that a supercoiled form is converted repeatedly into an identical form by permutation of the protofilament conformations from one to the next along the circumference in the direction opposite to its rotation, keeping the helical axis of the supercoil fixed in the same position and orientation. The hook protein and flagellin with very different structural characteristics form tubular structures with basically the same architecture and helical symmetry. Presumably, flagellin subunits are exported through the narrow central channel in a partially unfolded state. The cavity appears to have the right size to accommodate only one flagellin subunit at a time, allowing its refolding without aggregation with other subunits.

Formation of complex biological structures such as cells and cellular organelles is based on self-assembly of component macromolecules such as proteins and nucleic acids either in solution or within lipid membranes. The self-assembly processes are mostly driven by precise recognition of template structures by assembling molecules. The bacterial flagellum is one of the typical examples of this process. The well-established abilities of biological macromolecules to self-organize into complex three-dimensional architectures therefore give us a wonderful opportunity to use them in nanotechnology applications.

13. Bacterial toxins

Bacterial toxins were the first bacterial virulence factors to be identified and were laso the first link between bacteria and cell biology, i.e., the ―Cellular microbiology‖ was in fact naturally born a long time ago with the study of toxins, and recently has expanded to include study of many other aspects of prokaryotic cell –eukaryotic cell interactions.

Bacterial toxins have a target in most compartment / structures of eukarytic cells; Toxins are divided into three main categories: a) those that exert their toxicity by acting of the surface of target cell; b) those that have an intracellular target and hence need to cross the cell membrane ( these toxins need at least two active domains, one to cross the eukaryotic cell membrane and the other to modify the toxin target; and those c) that are directly delivered by the bacterial cell into eukaryotic cell.

Bacterial toxins target structures / processes:

Toxin cell surface interaction: Group 1: toxins act by binding receptor on cell membrane and sending a signal to the cell; Group 2: toxins act by forming pores in the cell membrane, per turbing permeability barrier; Group 3: toxins are A/B toxins, composed of a binding domain (B subunit) and an active effectordomain(A subunit). Following receptor binding, the toxins are internalized and located in , from which the A subunit can be transferred directly to the cytoplasm by using a pH –dependent conformatinal change, or can be transported to the Golgi apparatus / ER, from which the A subunit is finally released; Group 4: toxins are injected directly from the bacterial cell into the target cell by a contact dependent secretion system.

Production / chemical level:

The cell-associated toxins are referred to as endotoxins and the extracellular diffusible toxins are referred to as exotoxins. Endotoxins are cell-associated substances that are structural components of bacteria. Most endotoxins are located in the cell envelope. In the context of this article, endotoxin refers specifically to the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) located in the outer membrane of Gram-negative bacteria. Although structural components of cells, soluble endotoxins may be released from growing bacteria or from cells that are lysed as a result of effective host defense mechanisms or by the activities of certain . Endotoxins generally act in the vicinity of bacterial growth or presence. Exotoxins are usually secreted by bacteria and act at a site removed from bacterial growth. However, in some cases, exotoxins are only released by lysis of the bacterial cell. Exotoxins are usually proteins, minimally polypeptides, that act enzymatically or through direct action with host cells and stimulate a variety of host responses. Most exotoxins act at tissue sites remote from the original point of bacterial invasion or growth. However, some bacterial exotoxins act at the site of pathogen colonization and may play a role in invasion

Toxins categorized according to mode of action: damaging cell membranes, inhibiting protein synthesis, activating second messenger pathways, inhibiting the release of neurotransmitters, or activating the host immune response.

Organism/toxin Mode of action Target

Damage membranes

Aeromonas Pore-former Glycophorin hydrophila/aerolysin

Clostridium Pore-former Cholesterol perfringens/

perfringolysin O Escherichia coli/ Pore-former Plasma membrane hemolysind Listeria monocytogenes/ Pore-former Cholesterol

listeriolysin O

Staphyloccocus aureus/ Pore-former Plasma membrane -toxin

Streptococcus Pore-former Cholesterol pneumoniae/

pneumolysin

Streptococcus pyogenes/ Pore-former Cholesterol

streptolysin O

Inhibit protein synthesis

Corynebacterium ADP- Elongation factor 2 diphtheriae/ ribosyltransferase

diphtheria toxin

E. coli/Shigella N-glycosidase 28S rRNA dysenteriae/

Shiga toxins

Pseudomonas ADP- Elongation factor 2 aeruginosa/ ribosyltransferase

exotoxin A

Activate second messenger pathways

E.coli

CNF Deamidase Rho G-proteins

LT ADP- G-proteins ribosyltransferase STd Stimulates guanylate guanylate cyclase cyclase receptor

CLDTd G2 block Unknown

EAST ST-like? Unknown

Bacillus anthracis/ Adenylate cyclase ATP edema factor

Bordetella pertussis/ dermonecrotic toxin Deamidase Rho G-proteins

pertussis toxin ADP- G-protein(s) ribosyltransferase Clostridium botulinum/ ADP- Monomeric G-actin C2 toxin ribosyltransferase

C. botulinum/C3 toxin ADP- Rho G-protein ribosyltransferase Clostridium difficile/

toxin A Glucosyltransferase Rho G-protein(s)

toxin B Glucosyltransferase Rho G-protein(s)

Vibrio cholerae/cholera ADP- G-protein(s) toxin ribosyltransferase

Activate immune response

S. aureus/

enterotoxins Superantigen TCR and MHC II

exfoliative toxins Superantigen (and TCR and MHC II serine protease?)

toxic-shock toxin Superantigen TCR and MHC II

S. pyogenes/pyrogenic Superantigens TCR and MHC II exotoxins

Protease

B. anthracis/lethal factor Metalloprotease MAPKK1/MAPKK2

C. botulinum/neurotoxins A-G Zinc-metalloprotease VAMP/synaptobrevin SNAP-25 syntaxin

Clostridium tetani/ Zinc-metalloprotease VAMP/synaptobrevin tetanus toxin

14. Viroids

Viroids: are infectious agents (common plant pathogen) characterised by these bazic markers: They are all single stranded RNA covalently closed molecules with extensive intramolecular base pairing ; a DNA-directed RNA polymerase makes both plus and minus strands; no proteins are encoded. Two groups (families) are known: Avsunviroids and Pospiviroids (more 30 species have been identified).

Replication: Avsunviroids replicate via a symmetric rolling circle mechanism, whereas Pospiviroids use an asymmetric mechanism (?). By this: the (+) infecting circular RNA strand of a viroid serves as a template to make a large linear multimeric (-) strand. RNA pol II is probably the enzyme which does this. Pospiviroids with an asymmetric replication pathway then make + RNA strand from this long linear molecule. A host RNAse activity cleaves the + strand into unit viroid lengths. This molecule is then ligated to form a circular viroid. In Avsunviroid replication the long - RNA strand is self cleaved by the associated ribozyme activity. The RNA circularizes to form a - circle. A second rolling circle event makes a long linear + strand which is again cleaved by the ribozyme activity. The short viroid RNA is then ligated to the circular form. Additionally it is speculated that Avsunviroids may replicate in whereas Pospiviroids replicate in the nucleus and . 3 enzymatic activities are required for viroid replication, an RNA polymerase, an RNAse and an RNA ligase.

General structure:

Origin: some features of viroids suggest they may have originated in the hypothetical prebiotic RNA world. They can possess a ribozyme activity.They are GC rich which would attenuatethe low fidelities of replication activities. They are circular and so do not require start and stop functions for replication. They move within a plant, probably in association with host proteins via the phloem vascular channels and plasmodesmata cell contact points. They seem to form a quasi-species population and can recombine. Viroids of a particular type are widespread in some areas and absent in others.

There are fundamental differences between the aforementioned (two) groups (families) of viroids, reflecting different origins. Probably there is more than one mechanism responsible for viroid pathogenesis. Recent evidence suggests that one pathway is due to viroid RNA activating a plant RNA activated protein kinase, PKR (analogous to the PKR enzyme activated by viral RNAs in mammalian cells). Protein synthesis is reduced and this causes pathogenic effects. In addtion, changes in the viroid genome can alter its virulence. This reflects the fact that any (silencing) siRNA produced would have less complementary base pairing with target mRNA. Moreover siRNA corresponding to sequences from viroid genomes have been isolated from infected plants. This evidence indicates that when viroids replicate via a double stranded intermediate RNA, they are targeted by a Dicer (see Chapter 6.) and cleaved into siRNAs that are then loaded into the RISC. The viroid siRNAs actually contain sequences capable of complementary base pairing with the plant's own messenger RNAs and induction of degradation or inhibition of translation is what causes the viroid pathogenesis.

Keywords for further reading

1. Cytoskeleton (yeast cytoskeleton, spindle pole body, )

2. Motor proteins (kinesins, , dyneins)

3. Signal Sequences-Navigated Protein Transport (translocon, Sec systém, vesicular transport, exocytosis, protein secretory pathway)

4. Apoptosis (apoptic cell death, caspases, pro- / anti- apoptic proteins)

5. RNA: structures and function (RNPs, posttranscriptional modifications, RNA-binding proteins)

6. RNA interference (Dicer, Risc, Dosha, Pasha, siRNA, miRNA)

7. Control of Gene Expression (operon concept, regulon, induction / repression, promoter, operator, regulatory proteins, catabolite repression, negative / positive regulation)

8. Conjugative Gene Transfer (conjugative plasmids, conjugative transposons, HGT)

9. DNA damane response (DNA repair, photoreactivation, excision repair, recombination repair)

10. Cell – Cell Signaling (signal molecules, cell response, signal transduction, )

11. Bacterial Chemotaxis (chemotaxis pathway, bacterial flagellum motor)

12. Bacterial cell motility (cell gliding, flagelar motility)

13. Bacterial toxins (soluble toxins, cytotoxins, endotoxins, exotoxins, superantigens)

14. Viroids ( viroids origin, Avsunviroids, Pospiviroids, ribozyme)