Figure A summary of spontaneous alterations likely to require DNA repair.

Transcription

1 DNA Damage Figure A summary of spontaneous alterations likely to require DNA repair. The sites on each nucleotide that are known to be modified by spontaneous oxidative damage (red arrows), hydrolytic attack (blue arrows), and uncontrolled methylation by the methyl group donor S-adenosylmethionine (green arrows) are shown, with the width of each arrow indicating the relative frequency of each event. (After T. Lindahl, Nature 362: , Macmillan Magazines Ltd.)

2 Deamination of Cytosine

3 Thyamine dimers

4 Nucleases Cleave nucleotide sequences DNases and RNases and non specific nucleases ss and ds specificity Exonucleases (remove nucleotide from the end) Endonucleases (recognize palindromic ds DNA sequences)

5 Restriction endonucleases Three types (I, II, and III) I and III require ATP Type II are used as common molecular biology tools

6 Type II restriction enzymes Recognize and cleave particular sequences For example, BamHI GGATCC 5 -N-N-N-N-G-G-A-T-C-C-N-N-N-N-3 3 -N-N-N-N-C-C-T-A-G-G-N-N-N-N-5

7 BamHI 5 -N-N-N-N-G-G-A-T-C-C-N-N-N-N-3 3 -N-N-N-N-C-C-T-A-G-G-N-N-N-N-5 5 -N-N-N-N-G G-A-T-C-C-N-N-N-N-3 3 -N-N-N-N-C-C-T-A-G G-N-N-N-N-5 sticky ends overhanging sequence

8 Why do bacteria have endonucleases? How do they avoid digesting their own DNA?

9 Overview DNA structure A, B, and Z DNA DNA intercelators and groove binders Thermal melting of DNA DNA tertiary structure DNA methylation DNA damage nucleases

10 Which of the following statements correctly describes B-DNA A. B-DNA is usually found in solutions of reduced water B. B-DNA displays a wider helix in comparison to Z-DNA C. B-DNA forms a grooved left-handed helix D. B-DNA has a helix shorter and wider than A-DNA

11 RNA

12

13

14 Arrangement in three dimensions

15

16 Beyond the Four Bases

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18 Elements of RNA Secondary Structure

19 Elements of RNA Tertiary Structure

20 Messenger RNA (mrna) Transcription product of DNA In prokaryotes, a single mrna contains the information for synthesis of many proteins In eukaryotes, a single mrna codes for just one protein, but structure is composed of introns and exons

21 Eukaryotic mrna 5 Cap A phosphate is released by hydrolysis. The diphosphate 5 end then attacks the α-phosphorus atom of GTP to form a very unusual 5-5 triphosphate linkage. This distinctive terminus is called a cap. The N-7 nitrogen of the terminal guanine is then methylated by S-adenosylmethionine to form cap 0. The adjacent riboses may be methylated to form cap 1 or cap 2. Caps contribute to the stability of mrnas by protecting their 5 ends from phosphatases and nucleases. In addition, caps enhance the translation of mrna by eukaryotic proteinsynthesizing systems

22 Eukaryotic mrna poly A tail mrna molecule devoid of a poly(a) tail is usually a much less effective template for protein synthesis than is one with a poly(a) tail. enhances translation of mrna The half-life of an mrna molecule may also be determined in part by the rate of degradation of its poly(a) tail. enhances stability of mrna

23 Transfer RNA (trna) Recruits amino acid to the ribosome to synthesize protein Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem Many non-canonical base pairs found in trna

24 Secondary Structure of trna R = amino acid

25 Tertiary Structure of trna 5 3

26 Noncanonical base pairing and unusual bases in trna

27 Ribosomal RNA Facilitate protein synthesis Ribosomes are about 2/3 RNA, 1/3 protein rrna serves as a scaffold for ribosomal proteins 23S rrna in E. coli is the peptidyl transferase catalytic! RIBOZYME

28 Secondary structure of rrna

29 Small nuclear RNA (snrna) Participate in splicing the hnrna to form the mature mrna snrnp Size of snrna(nucleotides) Role U1 165 Binds the 5 splice site and then the 3 splice site U2 185 Binds the branch site and forms part of the catalytic center U5 116 Binds the 5 splice site U4 145 Masks the catalytic activity of U6 U6 106 Catalyzes splicing These RNA molecules and proteins assemble to form the Splicesome

30 snrna catalysis

31 Hammerhead catalytic RNA Self cleaving RNA involved in replication of single stranded viroid (RNA infectious agents of plant disease)

32 Small interfering RNA (sirna) Variety of roles in biology most characterized is the interference of the expression (translation) of a specific gene RISC - RNA-induced silencing complexes

33 DNA & RNA Differences? Why is DNA 2'-deoxy and RNA is not? Vicinal -OH groups (2' and 3') in RNA make it more susceptible to hydrolysis DNA, lacking 2'-OH is more stable This makes sense - the genetic material must be more stable RNA is designed to be used and then broken down

34 Hydrolysis of Nucleic Acids RNA is resistant to dilute acid DNA is depurinated by dilute acid DNA is not susceptible to base RNA is hydrolyzed by dilute base

35 RNA World Chemical view: Abiotic nucleotide chemistry RNA-catalyzed biochemistry. Biochemical view: RNA-based life Protein/DNA-based life. Why RNA vs Peptide or DNA

36 How could protein synthesis work without protein and DNA?

37 Polypeptides would have played only a limited role early in the evolution of life because their structures are not suited to self-replication in the way that nucleic acid structures are. However, polypeptides could have been included in evolutionary processes indirectly. For example, if the properties of a particular polypeptide favored the survival and replication of a class of RNA molecules, then these RNA molecules could have evolved ribozyme activities that promoted the synthesis of that polypeptide. This method of producing polypeptides with specific amino acid sequences has several limitations. First, it seems likely that only relatively short specific polypeptides could have been produced in this manner. Second, it would have been difficult to accurately link the particular amino acids in the polypeptide in a reproducible manner. Finally, a different ribozyme would have been required for each polypeptide. A critical point in evolution was reached when an apparatus for polypeptide synthesis developed that allowed the sequence of bases in an RNA molecule to directly dictate the sequence of amino acids in a polypeptide. A code evolved that established a relation between a specific sequence of three bases in RNA and an amino acid. We now call this set of three-base combinations, each encoding an amino acid, the genetic code. A decoding, or translation, system exists today as the ribosome and associated factors that are responsible for essentially all polypeptide synthesis from RNA templates in modern organisms. The essence of this mode of polypeptide synthesis is illustrated in Figure 2.8. An RNA molecule (messenger RNA, or mrna), containing in its base sequence the information that specifies a particular protein, acts as a template to direct the synthesis of the polypeptide. Each amino acid is brought to the template attached to an adapter molecule specific to that amino acid. These adapters are specialized RNA molecules (called transfer RNAs or trnas). After initiation of the polypeptide chain, a trna molecule with its associated amino acid binds to the template through specific Watson-Crick base-pairing interactions. Two such molecules bind to the ribosome and peptide-bond formation is catalyzed by an RNA component (called ribosomal RNA or rrna) of the ribosome. The first RNA departs (with neither the polypeptide chain nor an amino acid attached) and another trna with its associated amino acid bonds to the ribosome. The growing polypeptide chain is transferred to this newly bound amino acid with the formation of a new peptide bond. This cycle then repeats itself. This scheme allows the sequence of the RNA template to encode the sequence of the polypeptide and thereby makes possible the production of long polypeptides with specified sequences. The mechanism of protein synthesis will be discussed in Chapter 29. Importantly, the ribosome is composed largely of RNA and is a highly sophisticated ribozyme, suggesting that it might be a surviving relic of the RNA world.