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Home , GC box, Inducer, Pribnow box, Rho factor, Structural gene

Chapter 31

1. The role of RNA in synthesis 2. RNA polymerase 3. Control of transcription in 4. Posttranscriptional processing Three major classes of RNA

All participate in protein synthesis:

• Ribosomal RNA, rRNA 1. Transfer RNA, tRNA 2. Messenger RNA, mRNA

All are synthesized from DNA by transcription Historically DNA found in cell nucleus, but RNA found in cytosol (1930), microscopy and cell fractionation Concentration of cytosolic RNA-Protein particles correlate with protein synthesis - is site of protein synthesis - was later identified as In eukaryotes, DNA is never in association with protein synthesis (Ribosome) Incorporation of radiolabelled amino acids occurs in association with RNA-Protein particles Structure of DNA revealed possible copy mechanism Central Dogma

DNA

RNA

Protein 1. The role of RNA in protein synthesis

Studied by following enzyme induction

Bacteria vary the synthesis of certain enzymes depending on environmental Conditions

Enzyme induction occurs as consequence of mRNA synthesis Enzyme induction

E. coli can synthesize ˜4300 polypeptides

But enormous variation in abundance of specific polypeptides: Ribosomal protein: 10’000 copies/cell Regulatory protein: <10 copies/cell

Housekeeping enzymes, constitutive Adaptive, inducible enzymes Lactose-metabolizing enzymes are inducible

E. coli is initially unable to metabolize lactose, but starts to induce the corresponding enzymes: lactose permease, for uptake β-galactosidase, for splitting lactose

Few copies -> >10% or , 1000-fold Triggering substance: , allolactose or IPTG

The induction kinetics of β- galactosidase in E. coli The E. coli lac

Structural Z, β-galactosidase Y, lactose permease A, thiogalactisodase transacetylase

Control site Regulatory P, I, inducer O, operator can transmit genes via conjugation

Conjugation allows for bacterial

Constitutive mutation: induced even without inducer -> mutation in I, distinct but closely linked to structural genes

PaJaMo experiment, 1956, Arthur Pardee Francois Jacob, Jacques Monod Bacterial conjugation F– cell acquires an F factor from an F+ cell

Bacterial conjugation: Transfer of genetic material Between a donor cell, F+, and recipient, F-

Ability to conjugate/mate is encoded on a plasmid, F factor (fertility) F+ cell are covered with F pili Allows binding to F- cell surface

Formation of cytoplasmic bridge and transfer of genetic information Converts F- to F+ Transfer of the bacterial chromosome from an Hfr cell to an F– cell and its subsequent recombination with the F– chromosome

F factor can spontaneously integrate into the genome -> Hfr strain (high frequency of recombination)

-> transmission of genomic information upon mating: in fixed order time dependent (90min) Merozygote, partially diploid Recombination and integration The PaJaMo experiment

Mate Hfr I+Z+ to F- I-Z- in absence of inducer Monitor β-gal activity over time

Induction after 1h, cessation upon 2h -> Z+ in I- cells leads to constitutive induction, cessation upon transfer of I gene -> I gene codes for diffusible of Z, F- is resistant to T6 and streptomycin Messenger RNA

Second type of constitutive mutation Oc, operator constitutive, maps between I and Z genes In merozygote F’ Oc Z- / F O+ Z+, β-gal inducible But in Oc Z+ / F O+ Z- constitutive synth of β-gal

O Can control Z only when on same chromosome !!! -> cis acting control I is trans acting factor

Proteins are synthesized in two stages: 1. DNA is transcribed in mRNA 2. mRNA is translated into protein

This model explains behavior of lac system Messenger RNA

In the absence of inducer, I binds to O and represses synthesis of structural genes Z, Y, A

On binding inducer, repressor dissociates from O, permitting transcription and subsequent

Operator-Repressor-Inducer system represents a molecular switch

Oc is constitutive because repressor cannot bind, Cis-acting element

Coordination of all 3 protein by single polycistronic mRNA transcript, cistron mRNAs have their predicted properties

Kinetic of enzyme induction -> mRNA has to be rapidly synthesized and degraded, short half- rRNA turnover is slower, comprises 90% of cellular RNA The distribution, in a CsCl density gradient, of 32P-labeled RNA that had been synthesized by E. coli after T4 phage infection The hybridization of 32P-labeled RNA produced by T2-infected E. coli with 3H-labeled T2 DNA 2. RNA Polymerase

RNA polymerase is responsible for the DNA-directed synthesis of RNA (1960), dNTP and DNA-dep.

E. coli RNAP hplpenzyme, 459kD, αββ'ωσ subunit composition, sigma σ70 unit dissociates from core once RNA synthesis has been initiated

RNAP functions: 1. Template binding 2. RNA chain initiation 3. Chain elongation 4. Chain termination Components of E. coli RNA Polymerase Holoenzyme Electron micrograph of E. coli RNA polymerase (RNAP) holoenzyme attached to various promoter sites on T7 DNA Template binding o RNA synthesis is initiated only at specific sites on the DNA template o RNAP binds to its initiation sites at sequence elements called promoter, these are recognized by (K ≈10-14M) o Promoter, ca. 40bp element, located 5’ of structural gene, first base in RNA is +1, initiation site o If RNAP bound to promoter -20 to +20 are DNaseI protected o Consensus promoter sequence, hexamer centered at - 10 = , TATAAT, plus additional element at - 35 sequence in between not important, but distance o +1 is either A or G The sense (nontemplate) strand sequences of selected E. coli promoters

Rate of transcription varies 1000-fold, correlates with strength of promoter to bind RNAP Initiation requires formation of an open complex

RNAP binding alters accessibility of bases towards methylating agents (dimethyl sulfate, DMS), DMS- footprinting -> holoenzyme binds to only one side/face of DNA and melts DNA Chain initiation

+1 is purine, A > G Initiation reaction: pppA + pppN -> pppApN + Ppi Unlike DNA replication, RNA initiation does not need a primer

Crab claw shape of Taq RNAP, pincers formed by β and β ’ with a cavern between the two pincers

Prokaryotic initiation is inhibited by rifamycin B, Steroptomyces mediterranei, rifampicin commercial gram- positive antibiotic (also tubeculosis), does not block promoter bdg or elongation, but only initiation Structure of Taq RNAP core enzyme Chain elongation 5’ -> 3’ or 3’ -> 5’ growth ?

Label with γ[32P]GTP, chase with cold GTP If 5’->3’, RNA is permanently labelled If 3’->5’, RNA is unlabelled Transcription supercoils DNA

Elongation requires opening of dsDNA, bubble 2 models: RNAP swirls around DANN -> transcript would wrap DNA rotates -> DNA must be tethered Transcription occurs rapidly and accurately

In vivo rate is 20-50nt/sec, entirely processive, no exo- nucleolytic correction like in DNA pol. One mistake per 10’000nt, tolerable because: 1. Genes are repeatedly transcribed 2. Genetic code is redundant -> high prob. of silent mut. 3. Aa substitutions in proteins are often tolerated 4. Large portion of transcript is non-coding () Intercalating agents inhibit both RNA and DNA polymerase

Actinomycin D intercalates into DNA and RNA, inhibits transcription and replication Chain Termination

EM specific sites of termination, two common features in E. coli: 1. Series of 4-10 consecutive A-Ts, As on template 2. G + C-rich palindromic region upstream of A-Ts A hypothetical strong (efficient) E. coli

The stability of terminator G+C hairpin + weak base pairing Between RNA polyU and DNA template ensure termination Termination often requires the assistance of

50% of termination sites lack cis-acting terminator sequences, but require protein factor, Rho

in vivo transcripts often shorter than in vitro Rho is hexameric, 419Aa activity Recognition sequence on RNA transcript Eukaryotic RNA polymerase

3 distinct types of RNAP 1. RNAP I, in nucleoli, makes rRNA 2. RNAP II, in nucleoplasm, makes mRNA precursors 3. RNAP III, nucleoplasm, 5S rRNA, tRNA, small RNAs

Up to 600kD, up to 12 subunits, 5 of these present in all 3 RNAP types

RNAP II has extraordinary C-terminal domain, CTD 52 repeats of PTSPSYS, 50 Ser are phosphorylated Transcription is only initiated if CTD is unphosphorylated Elongation occurs only if CTD is phosphorylated Phosph. Converts initiation complex to elong. compl. RNA Polymerase Subunitsa X-Ray structure of yeast RNAP II that lacks its Rpb4 and Rpb7 subunits

Resembles Taq RNAP crab claw like shape Cutaway schematic diagram of the transcribing RNAP II elongation complex

On DNA binding, 50kD clamp swings out -> processivity Amatoxins specifically inhibit RNA polymerase II and III

Poisonous mushroom, Amanitia phalloides Responsible for majority of fatal mushroom poisonings Toxin, bicyclic octapeptide, amatoxins, α-amanitin Tight 1:1 complex with RNAP, K 10-8M Act slowly, death after a few days -> turnover of RNA Mammalian RNAP I has a bipartite promoter

Numerous rRNA genes have essentially identical sequence and promoter But unlike RNAP II and RNAP III, RNAP I promoters are Species specific !!

Core promoter -31 to +6 and upstream element (-187 to -107) RNAP II promoters are complex and divers

Euk RNAP II promoters are more complex than their prokaryotic homologues

GC-box upstream of constitutive genes Selectiveley expressed genes often contain TATA box (-27 to -10), resembles -10 of prok. Genes

Mutation sin TATA box cause heterogeneity in initiation

CCAAT box, -70 tp -90, i.e. in globin genes The promoter sequences of selected eukaryotic structural genes Enhancers are transcriptional activators that can have variable positions and orientations

Promoter element that act in both orientation and distance independent = enhancers

Can act from several kb, in euk. Viruses or structural genes

Required for full activity of promoter

Recognized by specific transcription factors -> DNA loop Stimulate entry of RNAP II on promoter

Mediate much of selective RNAP III promoters canbe located downstream from their transcription start site

RNAP III promoters can be within the gene’s transcribed region !! 5S rRNA 3. Control of Transcription in Adaptation to environmental changes takes only minutes because transcription and translation in prokaryotes are coupled (euk. takes hours) mRNAs are degraded in 1-3min Promoters

Genes that are transcribed at high rates have efficient promoters

Lac I is transcribed at 10 copies/cell Gene expression can be controlled by a succession of sigma factors

Cell development and differentiation involves the temporally ordered expression of specific sets of genes according to a genetically specified program

For example phage infection in prokaryontes 1. Expression of early genes 2. Expression of middle / late genes

One way of regulation: cascade of sigma factors that recognize the respective promoters lac Repressor I: Binding

1966 isolation of repressor based on binding to radio- Labelled IPTG, protein low abundance (0.002%)

Tetramer, 360 Aa, binds DNA K = 10-6M, promoter 10-13M Trypsin cleavage releases two domains. N-term. binds DNA rest binds IPTG

Protein scans DNA to bind to promoter (on rate is greater than diffusion limited process). lac operator has a nearly palindromic sequence

Repressor protein used to “fish” binding DNA sequence

Lac I binds to 26bp element with nearly 2-fold symmetry lac repressor prevents RNA polymerase from forming a productive initiation complex

RNA polymerase binds +20 to -20 Operator occupies +28 to -7 -> lac operator and promoter overlap Binding of repressor obstructs RNAP binding Catabolite Repression: An example of gene activation

Glucose is the metabolite of choice !!! In its presence, no other C-source is being metabolized, >100 enzymes are repressed (arabinose, galactose, lactose) = catabolite repression

Prevents wasteful duplication of energy producing enzymes The kinetics of lac operon mRNA synthesis following its induction with IPTG, and of its degradation after glucose addition cAMP signals the lack of glucose

cAMP is second messenger in animal cells In E. coli, cAMP greatly diminished in presence of glucose

Addition of cAMP to culture overcomes catabolite repression CAP-cAMP complex stimulates the transcription of catabolite repressed

cAMP binding protein = catabolite gene protein, CAP = cAMP protein, CRP

Homodimer of 210 Aa, undergoes large conformational change upon cAMP bdg.

CAP-cAMP binds lac operon and stimulates transcription -> positive regulator (unlike lac I, negative regulator)

Binds DNA and bends it 90°, contacts CTD of RNAP X-Ray structures of CAP–cAMP complexes Sequence-specific protein-DNA interactions

Genetic expression is controlled by proteins such as CAP, lac repressor

How do proteins bind to specific DNA sequences, how do they recognize base (pairing) ?

Base position in minor (5Å wide, 8Å deep groove is sequence independent, but varies in major groove ! -> protein / base recognition via major groove, 12Å wide, 8Å deep The helix-turn-helix is a common DNA recognition element in prokaryotes

Cap dimer’s two symmetrical F helices fit into two successive major grooves of B-DNA

CAP’s E and F helix form a helix-turn-helix (HTH) motif (supersecondary structure), similar to lac repressor, trp repressor

HTH is 20 Aa motif, helices cross at 120°

F helix in CAP is recognition helix, complex structural interactions (hydrogen bonds, salt bridges and van der Waals interactions), i.e. no simple Aa - Base code HTH-DNA interaction araBAD operon:positive and negative control by the same protein

Arabinose is not metabolized by human, but E. coli in our gut will metabolize this pentose

5 enzymes form an catabolite repressible araBAD operon

Control sites araI, araO1, araO2

Regulator: araC, homodimer 292Aa, N-term arabinose binding and dimrization domain, linker + C-term DNA bdg. domain Genetic map of the E. coli araC and araBAD operons Mechanism of araBAD regulation lac repressor II: structure

DNA loop formation is an important mechanism for transcriptional regulation

The lac repressor is a dimer of dimers, V-shaped Model of the 93-bp DNA loop formed

when lac repressor binds to O1 and O3

DNA loop is further stabilized by cAMP-CAP binding

Principal: Modular build up and break down of very high affinity complexes : Attenuation

Attenuation: Control mechanism to regulate amino acid biosynthetic operons

E. coli trp operon: five polypeptides, 3 enzymes, mediate the synthesis of trp from chorismate

Regulated by trp repressor, homodimer, 107Aa, bind L-trp -> binds trpO -> repression Trp acts as a Genetic map of the trp operon Tryptophan biosynthesis is regulated by attenuation trpE, first structural gene in trp operon is preceded by trpL, 162nt leader sequence

Availability if trp results in premature transcription Termination within trpL

Control element for this transcription termination = attenuator trpL contains 2 consecutive trp codons thereby couples Translation rate to formation of RNA secondary structure and transcription termination

Similar in his operon, ilv operon The alternative secondary structures of trpL mRNA The trp attenuator’s transcription termination is masked when trp is scarce Amino Acid Sequences of Some Leader Peptides in Operons Subject to Attentuation Regulation of rRNA synthesis: The stringent response

E. coli division 20 min, contains 70’000 -> must synthesize 35’000 ribosomes/20 min

Initiation of rRNA transcription : 1 /sec -> 1200 ribosomes/20min

⇒Seven distinct rRNA operons per chromosome + multiple replicating chromosomes

Coordination: rate of rRNA synthesis proportional to rate of protein synthesis

Molecular control of this coordination: Stringent Response (p)ppGpp mediates the stringent response o The stringent response is correlated with a rapid intracellular accumulation of two unusual : ppGpp and pppGpp = (p)ppGpp o Rapid decay when amino acids become available o relA- mutants exhibit no stringent response = relaxed control lack (p)ppGpp o (p)ppGpp inhibits the transcription of rRNA genes, but stimulates transcription of trp and lac operons o (p)ppGpp alters RNAP promoter specificity o Rel A, stringent factor: ATP + GTP <-> AMP + pppGpp o Active in association with ribosome engaged in translation but lack charged tRNAs o (p)ppGpp degradation by SpoT 4. Posttranscriptional Processing

Primary transcripts - of eukaryotes - are not yet functional but undergo post-transcriptional modifications: 1. Exo- and endonucleolytic removal of nt 2. Appending nt at 3’ and 5’ ends 3. Modification of specific nucleosides Messenger RNA processing: caps, tails, and splicing

In eukaryotes: primary transcripts are made in the cell nucleus, but translation takes place in the cytosol

Primary transcripts are processes on their transport way to the cytosol Eukaryotic mRNAs are capped

Cap: 7-methylguoanosine is joined at 5’ nucleoside via 5’-5’ triphosphate bridge

Cap defines eukaryotic translation start site

Addition requires: 1. RNA triphosphatase 2. Capping enzyme 3. Guanine-7-methyltransf. 4. 2’-O-methyltransf. Eukaryotic mRNAs have poly(A) tails

Unlike prokaryotic mRNAs, eukaryotic mRNAs are always monocistronic.

Termination process in imprecise -> heterologous 3’ ends, but mature mRNAs have well defined 3’ends with tails of ~250 polyAdenosine nucleotides

Appended by two reactions: 1. Cleavage of (heterologous) 3’ end, ~20nt past AAUAAA sequence; CFI, CFII 2. Poly(A) polymerase, PAP

Poly(A) tail gets shorter as mRNA ages Mature mRNAs lack poly(A) tail Eukaryotic genes consist of alternating expressed and unexpressed sequences

Primary transcripts are heterogenous in size and much larger than mature mRNAs !! ->heterogenous nuclear RNA (hnRNA) mature to mRNAs by excision of internal sequences (pre-mRNA) Intervening sequence = intron (~1500nt, average 8/gene) Expressed sequence = (~300nt) Largest gene, titin 29’926 Aa, 234 , 17kb exon are spliced in a two-stage reaction

Gene splicing must be precise to maintain the reading frame !! The at the exon–intron junctions of vertebrate pre- mRNAs

Invariant GU at intron 5’ boundary, AG at 3’ boundary The sequence of transesterification reactions that splice together the exons of eukaryotic pre-mRNAs Types of Introns Exons are spliced in a two-stage reaction (2)

1. Formation of 2’,5’-phosphodiester bond between adenosine in intron and 5’ phosphate -> intron assumes lariat structure 2. Free 3’-OH of 5’ exon generates phosphodiester with 3’ exon, -> releasing the intron lariat

Intron lariat is then debranched, and degraded

Note. No free energy input ! Some eukaryotic genes are self-spliced

Today we know 8 distinct types of introns:

Group I introns, nuclei, mitochondria, chloroplasts Tetrahymena, ciliate, no protein required for splicing, RNA only + guanosine

Self-splicing RNA = ribozyme Group II introns Mitochondria of fungi and plants Self splicing, lariat intermediate but no external

Spliceosome is an RNA-protein complex that mediates splicing of normal pre-mRNA, evolved from group primordial self-splicing RNA, Protein thought to be important for fine-tuning of ribozyme structure,

Similar, RNA of ribosome has catalytic activity

=> RNA world hypothesis The self-splicing group I intron from Tetrahymena thermophila Hammerhead ribozymes catalyze an in-line nucleophilic attack Simplest and best characterized ribozymes Embedded in the RNA of certain plant viruses Termed hammerhead enzyme due to structural resemblance

Enzyme green Substrate blue Cleavage site red Splicing of pre-mRNAs is mediated by snRNPs in the spliceosome o How are splicing junctions recognized and how are the two exons joined ? o Eukaryotes contain many 60-300nt nuclear RNAs, termed small nuclear RNAs, snRNA o Form RNA-protein complexes termeds small nuclear ribonucleoproteins, snRNPs o U1-snRNA (U-rich) is complementary to 5’ splice site, recognizes this splice site o Splicing takes place in 45S particle, spliceosome, which brings together pre-mRNA and snRNPs, 5 RNAs, ~65 proteins, ATP-dep., U2-,U4-, U5-, U6-snRNPs Figure 31-55 The X-ray structure of the catalytic pocket in the hammerhead ribozyme’s kinetically trapped intermediate. 4 6 2 1 e g a P An electron micrograph of spliceosomes in action A schematic diagram of six rearrangements that the spliceosome undergoes in mediating the first transesterification reaction in pre-mRNA splicing

1. Exchange of U1 for U6 in base pairing to 5’ splice site 2. Exchange of BBP for U2 in binding to branche site 3. Intramolecular rearrangement in U2 4. Disruption of pairing between U4 and U6 5. Disruption of a second stem between U4 and U6 6. Disruption of a stem-loop in U2 Splicing also requires the participation of splicing factors o Variety of proteins known as splicing factors that are not part of the spliceosome also participate in the splicing reaction o Branche point binding protein, BBP (=SF1, U2AF) o SR proteins (Ser, Arg-rich) and members of the heterogenous nuclear ribonucleoprotein family (hnRNP), contain RRM domain (RNA Recognition Motif), hnRNP are highly abundant o Exon skipping does not normally occur, splicing occurs orderd in 5’->3’ direction, cotranscriptional Structure of the RNA binding portion of human branch point-binding protein (BBP) in complex with its target RNA Spliceosomal structures o All 4 snRNPs involved in pre-mRNA splicing contain the same snRNP core protein, which consist of 7 Sm proteins (react with autoantibodies from patients with systemic erythematosis), named B/B’, D1, D2, D3, E. F, and G protein o Each of these Sm proteins contain two conserved segments, Sm1 and Sm2 separated by a variable linker o The seven Sm proteins bind to conserved RNA sequence, the SM RNA motif, occurs in U1-, U2-, U4, and U5-snRNA o Form heptameric ring, central hole positive charged allows passage of ss RNA but not ds RNA o U1-snRNP consist of 10 proteins, 7 Sm proteins and 3 U1 specific factors A model of the snRNP core protein The electron microscopy-based structure of U1-snRNP at 10 Å resolution

The predicted secondary structure of U1-snRNA The molecular outline of U1-snRNP Significance of gene splicing o Why are there introns ? o Introns are rare in prokaryotic structural genes o Uncommon in yeast, 239 introns in 6000 genes o Abundant in higher eukaryotes o lack introns o Unexpressed sequences constitute 80% for a typical vertebrate structural gene o Molecular parasites (junk DNA) ? o Evolution of complex spliceosome must have been advantageous over elimination of split genes o intron/exon organization is: - An advantage for rapid evolution of new proteins - Allows gene function tuning through alternative splicing Many eukaryotic proteins consist of modules that also occur in other proteins o Example, LDL-receptor 839 Aa, 45kb gene, 18 exons, most encode specific functional domains, 13 of these segments/domains have homology with domains found in other proteins => modular construction of the LDL receptor => modular construction is found in many other proteins that are composed of re-utilized domains (i.e. signal transduction, SH2, SH3 domains etc.) Alternative splicing greatly increases the number of proteins encoded by eukaryotic genome o The expression of numerous cellular genes is modulated by the selection of alternative splice sites o Example rat α-tropomyosin gene encodes 7 tissue specific variants of the muscle protein Alternative splicing o Occurs in all metazoans o Human genome only 30’000 genes but estimated 50’000 - 140’000 structural genes o Entire functional domains or single amino acids can be altered in proteins through alternative splicing - soluble or membrane bound - can be phosphorylated by a specific kinase or not - subcellular localization - whether enzyme binds a specific allosteric effector - affinity of receptor - ligand interaction o Selection of alternative splice sites is developmental and tissue specific (regulation in space and time) Selection of alternative splice sites Best understood for Drosophila sex-determination genes

no TRA protein functional TRA protein (repression of splice site by Sxl)

Male-specific DSX protein -> represses female-specific genes Female-specific DSX protein -> represses male-specific genes (activation fo splice site) AU-AC introns are excised by a novel spliceosome

o Small fraction of introns (~0.3%) have AU rather than GU at their 5’ends and AC rather than AG at 3’ But are excised via lariat intermediate Are splice by a AU-AC spliceosome with U5 sn RNP in common but specific U11, U12 and U4atac-U6atac Trans-splicing

o Trans-splicing, joining of two separate RNA molecules - Observed in Trypanosomas, all mRNAs have same 35nt leader, but this leader is not present in the corresponding genes - Splice leader (SL) RNA, transcribed from an independent gene - Trans-splicing reaction resembles spliceosome-mediated cis-splicing - But Y-shaped rather than lariat intermediate The sequence of transesterification reactions that occurs in trans-splicing RNA can be edited by the insertion or deletion of specific nucleotides o Certain RNA differ in sequence from their corresponding genes Examples: C->U and U->C changes Insertion or deletions of U Insertion of multiple G or C residues o Most extreme case in mitochondria of Trypanosomes involves addition and removal of hundreds of U’s to and from 12 otherwise untranslatable mRNAs o RNA editing, violates central dogma ? Because not template-based o Discovery of guide RNAs (gRNAs), 50-70nt, 3’ oligo(U) tail A schematic diagram indicating how gRNAs direct the editing of trypanosomal pre-edited mRNAs

RNA editing occurs on ~20S RNP, editosome gRNA is used as template to “correct” the mRNA Requires: 1. Endonuclease 2. Terminal uridylyltransferase 3. RNA ligase Trypanosomal RNA editing pathways RNA can be edited by base deamination o Humans express two forms of apolipoprotein B (apoB): - apoB-48, only made in intestinal tissue, functions in chylomicron transport, triacylglycerol to liver and periphery - apoB-100, made in liver, functions in VLDL, IDL, and LDL to transport cholesterol from liver to periphery o apoB-100 is 4536Aa large protein, apoB-48 consists of apoB-100 N-terminal 2152 residues but lacks C-term domain of apoB-100 that mediate LDL receptor binding o Both are expressed from the same gene, mRNAs differ in a single C->U change, resulting in stop codon (UAA) o Base change mediated by a protein, cytidine deaminase substitutional editing, also in glutamate receptor RNA interference o Noncoding RNAs can have important roles in controlling gene expression o Anti-sense RNA can block translation of a specific message Yet injection of sense RNA into C. elegans also blocks protein production o Added RNA interferes with gene expression = RNA interference, RNAi o 1998 Andrew Fire and Craig Mello, Nobelprice 2006 ds RNA is substantially more efficient in causing RNAi induced by only a few molecules -> catalytic rather than stoichometric A model for RNA interference (RNAi)

1. Trigger RNA is cut to 21-23nt oligos =small interf. RNA siRNA, with 2nt overhang at 3’ and 5’ phosphatemediated by RNase, Dicer 2. siRNA is transfered to multisubunit RISC complex, RNA- induced silencing, siRNA guides substrate specificity 3. RISC cleaves mRNA, which is then further degraded

RNAi requires that trigger dsRNA is copied, mediated by RNA- dependent RNA polymerase (RdRP)

Method of choice for knockout study A model for transitive RNAi

siRNA can prime for RdRP- catalyzed synthesis of secondary trigger dsRNAs which are diced to yield secondary siRNAs

May yield non-specific silencing, Transitive RNAi

RNAi may arose as defense against RNA viruses, inhibit movement of retrotransposons Ribosomal RNA Processing o Seven rRNA copies in E. coli genome, o polycistronic >5500nt transcript, containing 16S rRNA, 1-2 tRNAs, 23S rRNA, 5S rRNA, plus 1-2 more tRNAs at 3’ o Processing into mature rRNAs, cotranscriptional o Specific endonucleolytic cleavage by RNase III, RNase P, RNase E, RNase F o Secondary processing, trimming of 5’ and 3’ ends occurs while rRNA is already associated with ribosomal proteins The posttranscriptional processing of E. coli rRNA Ribosomal RNAs are methylated o During ribosome assembly, 16S and 23S rRNA is methylated at 24 specific nucleosides, SAM-dep. o N6,N6-dimethyladenine and O2-methylribose (protect from RNase degradation) Eukaryotic rRNA processing is guided by snoRNAs o rRNA transcription and processing takes place in nucleoli o Primary transcript is ~7500nt 45S RNA that contains 18S, 5.8S, 28S rRNAs separated by spacer sequences o Specific methylation (106 sites in humans) o Conversion of U to pseudouridine (95 in humans) o Subsequent cleavage and trimming analogous to prok. o How are methylation sites recognized/targeted ? o Pre-rRNA interact with small nucleolar RNAs, snoRNAs ( ~200 in mammals), intron-encoded The organization of the 45S primary transcript of eukaryotic rRNA Transfer RNA processing o tRNA, ~80nt, cloverleaf secondary structure, large fraction of modified bases o E. coli chromosome contains 60 tRNA genes, some are components of rRNA operons o Primary transcript contains 1-5 tRNA copies, excision and trimming similar to rRNA processing o Many tRNAs contain introns o CCA trinucleotide to which the amino acid is appended is postracriptionally added, tRNA nucleotidyltransferase o RNase P generates 5’ ends of tRNAs, contains 377nt RNA component, is catalytic subunit A schematic diagram of the tRNA cloverleaf secondary structure Figure 31-74a The structure of the RNA of B. subtilis RNase P. (a) Predicted secondary structure with specificity domain drawn in various colors and catalytic domain is black. 8 7 2 1 e g a P Figure 31-74b The structure of the RNA of B. subtilis RNase P. (b) The X-ray structure of the specificity domain in which its various segments are colored as in Part a. 8 7 2 1 e g a P The posttranscriptional processing of yeast tRNATyr