Topic: MOLECULAR BASIS OF INHERITANCE. REALISATION OF HEREDITARY INFORMATION Plan 1. History of discovering of nucleic acids. 2. Evidences of genetic role of nucleiс acids. 3. Characteristics of DNA and RNA. 4. DNA replication. Self-correction and reparation of DNA. 5. Gene as a unit of genetic information. Structure of pro- and eukaryotes genes. 6. Flow of information in a cell. Transcription, processing, splicing. 7. Genetic code and its characteristics. 8. Translation and posttranslational modification of protein. Gene expression. Recommended literature:
1. Medical Biology : text-book / Bazhora Yu. I., Bylyk R. Ye., Chesnokova M. M. et al. – 2nd ed., revised and updated. – Vinnytsia : Nova Knyha, 2019. – 448 p. 2. Biological peculiarities of the human vital functions. Training text-book on Medical biology (module I) for students of medical and stomatological faculties / S.I. Dubinin, O.V. Ovcharenko, O.V. Klepets et al. – Poltava, 2019. – 79 p. 3. Lazarev K.L. Medical Biology: Text-book. – Second edition. – Simferopol: IAD CSMU, 2003. – 592 p. 4. Medical biology: The study guide of the practical classes course / O.V. Romanenko, O.V. Golovchenko, M.G. Kravchuk, V.M. Grinkevych. – Edited by O.V. Romanenko. – K.: Medicine, 2008. – 304 p. 5. Bekish O.-Y.L. Medical biology: Textbook for students of higher educational establishments. – Vitebsk: VSMU Press, 2003. – 346 p. 6. Green N.P., Stout G.W., Taylor D.I. Biological Science. – Cambridge, 1994. History of discovering of nucleic acids 1868 – Friedrich Miescher isolated some nonprotein phosphorus- containing substance in nuclei of pus cells (leykocytes) and salmon sperms, which called nuclein. 1881 – Albrecht Kossel investigated, Friedrich Miescher Richard Altmann 1844 – 1895 that nuclein is composed of four 1852 – 1900 bases – 2 purines and 2 pyrimidynes. 1889 – Richard Altmann found that nuclein has acidic properties and renamed it nucleic acid. 1909 – Phoebus Levene discovered that DNA is made of 3 basic components – a sugar, an acid and an Albrecht Kossel Phoebus Levene 1853 – 1927 organic base. 1869 – 1940 Phenomena, that prove the genetic role of nucleic acids 1. Transformation – is the inclusion of alien DNA into the genome of the host cell, leading to changes in its structural and functional properties (Griffith, 1928; Avery et al., 1944). 2. Transduction – is a form of horizontal gene transfer in which the transmission of genetic material from one cell to another occurs by a virus, which leads to changes in hereditary properties (Lederberg, Zinder, 1952). 3. Conjugation – is the transfer of genetic material between bacterial cells by a bridge-like connection between two cells (Lederberg, Tatum, 1947; Davis, 1950). Transformation 1928 – Fredrick Griffith discovered that genetic information could be passed from one bacteria to another – transforming principle. 1928 – Griffith’s experiment on mice with virulent and non-virulent strains of Streptococcus pneumoniae
Conclusion: there is some chemical factor in a cell that can genetically transform another cell. 1944 – Avery-MacLeod-McCarty Experiment
• 1944 Avery-MacLeod-McCarty Experiment
Conclusion: DNA is transforming factor. 1952 – Lederberg-Zinder experiment on transduction
gene T+ Joshua Lederberg 1925 – 2008
lysed cells
bacteriophage bacteriophage with gene T+
fine-pore filter, permeable for bacteriophages only
Strains of Salmonella typhimurium: Norton Zinder 22A – non-synthesis tryptophan strain, 1928 – 2012 2A – synthesis tryptophan bacterial strain (T+) Experiments on conjugation
Conclusion: the contact between cells is required. Study of DNA structure 1947 – Erwin Chargaff – isolated DNA from different organisms and measured number of each base. Found the ratio of bases (Chargaff’s rules): • number of purines is equal to number of pyrimidines: (A+G) = (T+C), so (A+G) /(T+C) = 1, A = T and C = G. • (A+T) /(G+C) varies from species to species – Erwin Chargaff 1905 – 2002 the coefficient of DNA specificity. Study of DNA structure 1952 – Franklin & Wilkins – x-ray diffraction of crystalline DNA fiber. It was discovered that DNA molecule: • characterized by helical 3D-structure; • has a diameter of 2 nm; • makes a complete turn of the helix every 3.4 nm. Rosalind Franklin 1920 – 1958
Maurice Wilkins 1916 – 2004 Study of DNA structure
James Watson born 1928 1953 – Watson & Crick proposed a double helix DNA model: DNA is a double helix made of coiled around a central axis, two anti-parallel strands of nucleotides linked by hydrogen bonds between complementary base pairs. Francis Crick 1916 – 2004 Main facts about nucleic acids • store the information about cell growth and reproduction; • represented by two main types – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA); • are polymers consisting of monomers called nucleotides; • a nucleotide consists of: 1) a nitrogenous base, 2) a pentose sugar and 3) a phosphate group. The structure of nucleotide The properties of DNA: 1. Antiparallel strands, which run in opposite directions: • leading (or informative, template) strand is oriented in 5’ to 3’, • lagging strand – in 3’ to 5’. 2. Complementarity: two strands are joined together through complementary base pairing: A = T, C ≡ G. 3. Replication – process of doubling of DNA molecule. 4. Semi-conservatism: each of maternal strand serve as a template for new synthesized daughter strand. 5. Species specificity: the DNA content in cells of every species is constant and specific, defined by ratio (A+T) / (G+C). 6. Spatial structure: presence of from primary to quaternary chemical structures and specific (A to Z) conformations of DNA molecule. 7. Collinearity: accordance of nucleic acids’ codons to amino acids of polypeptides. 8. Reparation – ability to identify and correct damages of DNA molecule. Complementarity DNA is made of four nucleotides: A, G, T, C, replaced by pairs. Only pyrimidine-purine pairing would produce the 2 nm diameter indicated by the X-ray data.
Thus, in every pair one purine base bonds with one pyrimidine base: Adenine always forms double hydrogen bonds with thymidine: A = T; Cytosine forms triple hydrogen bonds with guanine: C ≡ G. Semi-conservatism (Meselson, Stahl, 1950s) Base pairing allows to each strand to serve as a template for a new strand. Effects: New duplex is ½ parent template and ½ new DNA; Parental strands are not degraded. Species specificity of DNA (Chargaff,1947) The ratio(A+T) /(G+C) varies from species to species – the coefficient of DNA specificity: • (G+C) > (A+T) => GC-type of organisms: viruses, bacteria, fungi. • (A+T) > (G+C) => AT-type of organisms: higher plants, animals, human. Spatial structure of DNA:
1) primary – order of bases in polynucleotide sequence; 2) secondary – a double helix; 3) tertiary – supercoiling of molecule – the three- dimensional conformation of the backbone; 4) quaternary – interaction between DNA and protein. Spatial structure of DNA Primary structure – the sequence of bases along the pentose-phosphodiester backbone of DNA molecule, read from the 5’ to 3’ end. Spatial structure of DNA Secondary structure – the complex of two polynucleotide chains right twisted around common axis so that carbohydrate chain are outside the phosphate and nucleic bases directed inwards (Watson-Crick double helix).
A major factor stabilizing double helix is base pairing by hydrogen bonding. Spatial structure of DNA Tertiary structure – three-dimensional conformation of the backbone – the supercoiling – the coiling of the coil, or further coiling and twisting of DNA helix. Types of tertiary structure: linear: chromosomes of eukaryotes; circular: bacterial chromosomes, plasmids, some viruses.
Enzymes called topoisomerases, or gyrases can introduce or remove supercoils. Spatial structure of DNA There are different conformations of DNA double helix: A-DNA right-handed helix of 2.2 nm diameter 11 base pairs per turn of the DNA has not been found in vivo, except of bacterial spores B-DNA right-handed helix of 2 nm diameter 10 base pairs per turn of the helix physiological form Z-DNA left-handed double helix of 1.8 nm diameter 12 base pairs per turn may be involved in gene expression alternating purine and pyrimidine sequences. Spatial structure of DNA Quaternary structure – interaction between DNA and protein. DNA quaternary structure is used to refer to the binding of DNA to histones to form nucleosomes, and then their organisation into higher-order chromatin fibres. DNA quaternary structure varies over time, as regions of DNA are condensed or exposed for transcription. Replication – is a difficult biological process of reaction of matrix synthesis which provides doubling of DNA molecule.
• The process of DNA replication is a fundamental process used by all living organisms as it is the basis for biological inheritance. • This process is remarkably accurate, with only one error per billion nucleotides. • The DNA replication requires more then a dozen enzymes and other proteins, as well as energy consumption. • A human cell can copy its 6 billion base pairs and divide into two daughter cells in only a few hours. Okazaki fragments.
Summary of replication Proteins of DNA Replication DNA exists in the nucleus as a condensed, compact structure. To prepare DNA for replication, a series of proteins aid in the unwinding and separation of the double-stranded DNA molecule. • Тоpoisomerases alter the supercoiling of double-stranded DNA and facilitate the untwisting of supertwisted DNA. • Helicase breaks the hydrogen bonds between nitrogenous bases of two opposite strands of a double helix and then separates these strands, exposing two templates. • Single-strand binding (SSB) proteins bind to and stabilize the unwound single-stranded DNA until it can be used as a template. • Primase is an enzyme that synthesizes short RNA sequences called primers, which serve as starting points for DNA synthesis on 5’ end of the leading strand as well as each Okazaki fragment . • DNA-polymerase forms a new DNA chain in the 5’→3’ direction. • DNA-ligase links up the short DNA segments (known as Okazaki fragments), creating phosphodiester bonds.
Reparation – ability to identify and correct damages of DNA molecule.
RNA – ribonucleic acid – is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. The structure of RNA is similar to DNA with some key differences: Main types of RNA
Functions of nucleic acids 1. DNA is carrier of genetic information. The function is provided by the fact of existence of genetic code. 2. Reproduction and transfer of genetic information in the generations of cells and organisms. The function is provided by the process of DNA replication. 3. Realization of genetic information. The function is provided by the processes of transcription (DNA →mRNA) and translation (mRNA →polypeptide).
GENE – is a fragment of DNA molecule, which encodes the primary structure of protein / tRNA / rRNA or regulates the transcription of another gene. Formation of ideas about gene 1865 Gregor Mendel opened the laws of inheritance and concept about the inherited factors (genes). 1909 Wilhelm Johansen firstly used the term «gene». 1908 Archibald Edward Garrod proposed «one gene – one product» hypothesis. 1911 Тhomas Morgan worked out on «chromosomal theory»: a gene is an area of chromosome, responsible for the display of certain sign. 1926 in his book «Theory of gene» Morgan marked, that gene is a unit of mutation, recombination and function. 1940 George Biddle and Edward Tatum suggested a hypothesis «one gene is one enzyme». 1949 Linus Pauling and Vernon Martin Ingram established the role of genes in protein synthesis. 1955 Seymour Benzer determined the detailed structure of viral genes and coined the term «cistron» to denote functional subunits of genes. 1965 Charles Yanofsky proposed «one gene – one polypeptide» hypothesis. The structure of gene Parts of gene
Regulatory regions – Structural part (coding regulate transcription of regions) – encode proteins structural genes and all types of RNA
Promoter is a region to which RNA polymerase binds to initiate the transcription.
Terminator is a sequence at the end of a transcription unit that causes RNA polymerase to stop transcription. Exons – coding, informative fragments Operator is a sequence that interacts with a protein-repressor to regulate the expression of adjacent structural genes.
is a region of DNA that can be Enhancer Introns – noncoding, bound by protein (s) to increase the transcription. passive fragments
Silencer is sequence that reduces transcription by binding to repressor proteins. The lac operon model of prokaryotes gene expression In 1961 François Jacob and Jacques Monod described the genetic regulation of the lac operon in bacteria Escherichia colі. François Jacob 1920 – 2013 Operon is a group of genes with a single promoter (transcribed as a single mRNA), that are functioning as a single unit (encode proteins of the same metaboliс process).
The lac operon encode proteins that allow the bacteria to use lactose as an energy source. Jacques Monod 1910 – 1976 Negative regulation of lac operon expression
Regulated genes can be switched on/off depending on cell’s metabolic needs.
Gene expression is the multistage process by which information from a gene is used in the synthesis of a functional gene product – proteins or RNA. Transcription is the process in which a gene's DNA sequence is copied (transcribed) to make an RNA molecule. • Transcription takes place in a nucleus. • RNA polymerase is the main transcription enzyme. • Transcription begins when RNA polymerase binds to a promoter sequence near the beginning of a gene (directly or through helper proteins). • RNA polymerase uses one of the DNA strands (the template strand) as a template to make a new, complementary RNA molecule. Transcription of a gene takes place in three stages: initiation, elongation and termination.
1.Initiation. After RNA polymerase binds to the promoter, the DNA double helix unwinds and unzips, and the enzyme initiates RNA synthesis at the start point on the template strand. 2.Elongation. The RNA polymerase moves downstream, unwinding the DNA and elongating the RNA transcript in 5’→ 3’. The RNA transcript carries the same information as the non-template (coding) strand of DNA, but it contains the base uracil (U) instead of thymine (T). 3.Termination. The polymerase transcribes a short DNA terminator sequence, which signals the end of the transcription unit. The primary RNA transcript is released, and the enzyme detached from the DNA. In eukaryotes there is RNA Processing – a posttranscriptional process of mRNA maturation from precursor messenger RNA (pre-mRNA, primary transcript) to mature messenger RNA (mRNA). Events of processing: 1. Modification of pre-mRNA with forming protective 5’ and 3’ untranslated regions (UTR): A) Capping – modification of the head 5' end by adding of 7-methylguanosine- triphosphate – 5′ cap. B) Polyadenylation is an adding of up to 250 adenines to the 3' end of the mRNA – formation of poly-A tail (AAUAA is the poly-A processing signal). 2. Splicing – is a process of removing the non-coding sequences of genes (introns) from pre-mRNA and joining the protein- coding sequences (exons) together in order to enable further translation of mRNA into a protein. Alternative splicing
Alternative, or differential, splicing – is a regulated process where multiple proteins are encoded by a single gene via particular exons, parts of exons or introns are differentially joined or skipped. Hence, one gene may code for a large number of different products. Alternative splicing occurs as a normal phenomenon in eukaryotes, where it greatly increases the biodiversity of proteins that can be encoded by the genome. In humans, ~95% of multi-exonic genes are alternatively spliced. Genetic code is a system of rules by which information, encoded in genetic material (sequences of DNA or mRNA), is translated into amino acids sequences. The properties of genetic code: 1.Triplet nature: one amino acid is specified by three adjacent nucleotides, called codon, or triplet. 2.Universality: code is the same in all living organisms (except of some codons in mitochondrial DNA). 3.Collinearity: a linear arrangement of codons in mRNA determines a linear arrangement of amino acids in protein. 4.Degeneracy, or redundancy: most amino acids (18 of 20) are coded by more then one codon; codons, that specify the same amino acid, are called synonyms. 5.Code is specific (unumbiguous): every triplet codes a certain one amino acid. 6.Code is non-overlapping: each nucleotide is a part of one triplet only. 7.Code is commaless: there is no punctuation between adjacent codons. The table of genetic code Translation, or protein biosynthesis is the process during which a base sequence of nucleiс acids is translated into an amino acid sequence of polypeptide.
The most important components of protein biosynthesis: • mRNA • tRNA • ribosomes • amino acids • energy (ATP, GTP) • Mg2+ ions (are needed for association of ribosome subunits) • aminoacyl-tRNA synthetases enzymes. Ribosomes are organelles (in all cells) where proteins are synthesized. They are the translation factories, made of a large subunit and a small subunit. They consist of 2/3 rRNA and 1/3 protein. The ribosome has enzymes and ATP molecules to help it link one amino acid to another one. Ribosome has 3 main sites: P, E, A. • A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. • P site attached to the growing polypeptide. • E site is exit site where tRNA leaves the ribosome.
FOLDING = Posttranslational modification of proteins Functional types of proteins References: • https://slideplayer.com/slide/4643055/ • https://www.slideserve.com/qiana/structure-of-dna-1-primary-structure- 2-secondary-structure-3-tertiary-structure • https://slideplayer.com/slide/1522356/ • http://hobbieroth.blogspot.com/2018/03/chromatin-packing-and- molecular-biology.html • https://www.slideshare.net/ktanaka2/chapter-16-6621382 • https://www.slideshare.net/CWood01/dna-history-and-replication- review-presentation?next_slideshow=1 • https://pt.slideshare.net/Shababmehebub/dna-replication-56622894 • https://www.slideshare.net/namarta28/dna-damage-and-repair- 31989711 • https://www.khanacademy.org/science/biology/gene-expression-central- dogma/transcription-of-dna-into-rna/a/overview-of-transcription • http://nusep.com/ • https://slideplayer.com/slide/6303336/