Principles of Biochemistry Lecture 6 PDF - Spring 2024

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJ...

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJIMI DO NOT have a financial interest in commercial products or services. Lecture 6 DNA and RNA Structure Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 8: p. 263-283 NUCLEOTIDES AND NUCLEIC ACIDS Key topics: – Structure of double-stranded DNA – Structures of RNAs – Denaturation and annealing of DNA – Chemistry of nucleic acids; mutagenesis – Appendix: Structures of common nucleotides Biological function of nucleotides and nucleic acids NUCLEIC ACID STRUCTURE X-Ray crystallographic studies on DNA crystals X-ray diffraction pattern of DNA. The spots forming a cross in the center denote a helical structure. The heavy bands at the left and right arise from the recurring bases. 1953, Proposed the Double Helix Model for DNA Structure 1962, Nobel Prize in Physiol. & Med. shared by Crick, Watson, & Wilkins WATSON-CRICK MODEL OF B-DNA The original model proposed by Watson and Crick had 10 base pairs, or 34 Å (3.4 nm), per turn of the helix; Subsequent measurements revealed 10.5 base pairs, or 36 Å (3.6 nm), per turn. (a) Schematic representation, showing dimensions of the helix. (b) Stick representation showing the backbone and stacking of the bases. (c) Space-filling model. OTHER FORMS OF DNA (A AND Z DNA) Righthanded. Exists in poor water content (may not be physiologic) Watson-Crick (right-handed), most stable structure, observed in cells Left-handed (stretches exist in bacteria and eukarya, may play regulatory roles in cells or recombination) DNA can occur in different 3-D structures: The A, B, and Z forms of DNA Each structure shown has 36 base pairs. The riboses and bases are shown in yellow, the phosphodiester backbone is represented as a blue rope. COMPLEMENTARITY OF DNA STRANDS Two chains differ in sequence (sequence is read from 5’ to 3’) Two chains are complementary Two chains run antiparallel The complementary antiparallel strands of DNA follow the pairing rules proposed by Watson and Crick. The base-paired antiparallel strands differ in base composition: the left strand has the composition A3 T2 G1 C3; the right, A2 T3 G3 C1. They also differ in sequence when each chain is read in the 5′→3′ direction. Note the base equivalences: A = T and G = C in the duplex. REPLICATION OF GENETIC CODE Strand separation occurs first Each strand serves as a template for the synthesis of a new strand Synthesis is catalyzed by enzymes known as DNA polymerases Newly made DNA molecule has one daughter strand and one parent strand. “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” ―Watson and Crick, Nature, 1953 MESSENGER RNA: CODE CARRIER FOR THE SEQUENCE OF PROTEINS Is synthesized using DNA template Contains ribose instead of deoxyribose Contains uracil instead of thymine One mRNA may code for more than one protein Bacterial mRNA. Schematic diagrams show (a) Monocistronic mRNAs of bacteria. (b) Polycistronic mRNAs of bacteria. Red segments represent RNA coding for a gene product; gray segments represent noncoding RNA. In the polycistronic transcript, noncoding RNA separates the three genes. * In eukaryotes, alternative splicing can lead to the expression of several proteins from a single mRNA mRNA, together with transfer RNA (tRNA), transfers genetic information from DNA to proteins MANY RNAS HAVE COMPLEX 2-D AND 3-D STRUCTURES Typical right-handed stacking pattern of single-stranded RNA The bases are shown in yellow, the phosphorus atoms in orange, and the riboses and phosphate oxygens in green Secondary structure of RNAs. (b) The paired regions generally have an A-form right-handed helix Base-paired helical structures in an RNA as shown for the hairpin M1 RNA component of the enzyme RNase P of E. coli, with many hairpins. RNase P, which also contains a protein component (not shown), functions in the processing of transfer RNAs PALINDROMIC SEQUENCES CAN BE FOUND IN DNA Palindromes: words or phrases that are the same when read backward or forward: Example: rotator or nurses run 02/02/2020 -the first global palindrome day in 909 years. This is the only time such a date will occur this century. - The previous palindrome date in all formats came 909 years ago on 11/11/1111. - The next will come in 101 years on 12/12/2121 and after that there will not be another until 03/03/3030. A famous palindromes: "A Toyota's a Toyota" can continue as a palindrome forever, as in, "A Toyota's a Toyota's a Toyota..." PALINDROMIC SEQUENCES CAN FORM HAIRPINS AND CRUCIFORMS Intrastrand base pairing (one strand) Palindromes and mirror repeats. Palindromes are sequences of double-stranded nucleic acids with twofold symmetry. In order to superimpose one repeat (shaded sequence) on the other, it must be rotated 180˚ about the horizontal axis then 180˚ about the vertical axis, as shown by the colored arrows. A mirror repeat, on the other hand, has a symmetric sequence within each strand. Superimposing one repeat on the other requires only a single 180˚ rotation about the vertical axis. Palindromic DNA (or RNA) sequences can form alternative structures with intra-strand base pairing Intrastrand base pairing (two strand) RNA MOLECULES HAVE QUITE COMPLEX STRUCTURES General cloverleaf secondary structure of tRNAs A tRNA A Ribozyme An intron DNA DENATURATION Denaturation can be induced by high temperature, or change in pH Covalent bonds remain intact – Genetic code remains intact Hydrogen bonds are broken – Two strands separate Base stacking is lost – UV absorbance increases Denaturation may be reversible: annealing Reversible denaturation and annealing (renaturation) of DNA THERMAL DNA DENATURATION (MELTING) DNA exists as double helix at normal temperatures Two DNA strands dissociate at elevated temperatures Two strands re-anneal when temperature is lowered The reversible thermal denaturation and annealing forms the basis for the polymerase chain reaction DNA denaturation is commonly monitored by UV spectrophotometry at 260 nm Two DNAs with different nucleotide composition Heat denaturation of DNA: The denaturation, or melting, curves of two DNA specimens (with different nucleotide composition). The temperature at the midpoint of the transition (tm) is the melting point; it depends on pH and ionic strength and on the size and base composition of the DNA. FACTORS AFFECTING DNA DENATURATION The midpoint of melting (Tm) depends on base composition – High CG increases Tm Tm depends on DNA length – Longer DNA has higher Tm – Important for short DNA Tm depends on pH and ionic strength – High salt increases Tm Relationship between tm and the G+C content of a DNA DENATURATION OF LARGE DNA MOLECULES IS NOT UNIFORM AT rich regions melt at a lower temperature than GC-rich regions Partially denatured DNA: This DNA was partially denatured, then fixed to prevent renaturation during sample preparation. Single-stranded regions are readily distinguishable from double-stranded regions. The arrows point to some singlestranded bubbles where denaturation has occurred. The regions that denature are highly reproducible and are rich in A=T base pairs. TWO NEAR-COMPLEMENTARY DNA STRANDS CAN HYBRIDIZE DNA hybridization. Two DNA samples to be compared are completely denatured by heating. When the two solutions are mixed and slowly cooled, DNA strands of each sample associate with their normal complementary partner and anneal to form duplexes. If the two DNAs have significant sequence similarity, they also tend to form partial duplexes or hybrids with each other: the greater the sequence similarity between the two DNAs, the greater the number of hybrids formed. Hybrid formation can be measured in several ways. One of the DNAs is usually labeled with a radioactive isotope to simplify their detection and measurement. Denaturation and annealing is used for: Detection of a specific DNA molecule in complex mixture - radioactive detection - fluorescent DNA chips Amplification of specific DNA - polymerase chain reaction - site-directed mutagenesis Evolutionary relationships Antisense therapy ADENOSINE: A NUCLEOSIDE WITH PHYSIOLOGICAL ACTIVITY In general, nucleosides have no biological function other than to be part of nucleotides. Adenosine is an exception and functions in mammals as a local hormone and as a neuromodulator: As a local hormone, adenosine circulates in the bloodstream and act locally on specific cells to affect blood vessel dilation, muscle contraction, neurotransmitter release, metabolism of fat etc…(Example: during muscle contraction, [adenosine] increases à blood vessels dilate à blood flow increases and O2 and nutrients delivery to muscle increase) As a neuromodulator, adenosine is involved in sleep regulation: Extended wakefulness à increase in metabolism in the brain à [adenosine] increases à sleepiness. Caffeine, which promotes wakefulness is a competitive inhibitor of adenosine receptors TAUTOMERISM OF NUCLEOBASES MAY PLAY A ROLE IN DNA AND RNA MUTAGENESIS AND MISPAIRING Prototropic tautomers are structural isomers that differ in the location of protons Keto-enol tautomerism is common in ketones Lactam-lactim tautomerism occurs in some heterocycles (pyrimidines and purines) Both tautomers exist in solution but the lactam forms are predominant at neutral pH keto enol Tautomeric forms of uracil. The lactam form predominates at pH 7.0; the other forms become more prominent as pH decreases. This tautomerism is also found in Guanine. The other free pyrimidines and the free purines also have tautomeric forms, but they are more rarely encountered SPONTANEOUS MUTAGENESIS IN DNA Deamination Very slow reactions Large number of residues à The net effect is significant: 100 C ® U events /day in a mammalian cell Depurination N-glycosidic bond is hydrolyzed Significant for purines: 10,000 purines lost/day in a mammalian cell Cells have mechanisms to correct most of these modifications (see DNA repair) Some well-characterized nonenzymatic reactions of nucleotides. (a) Deamination reactions. (b) Depurination, in which a purine is lost by hydrolysis of the N-β-glycosyl bond. Loss of pyrimidines via a similar reaction occurs, but much more slowly. The resulting lesion, in which the deoxyribose is present but the base is not, is called an abasic site or an AP site (apurinic site or, rarely, apyrimidinic site). The deoxyribose remaining after depurination is readily converted from the β-furanose to the aldehyde form, further destabilizing the DNA at this position. THE CHEMICAL DIFFERENCES BETWEEN DNA AND RNA HAVE BIOLOGICAL SIGNIFICANCE Two fundamental chemical differences between DNA fand RNA: 1. DNA contains 2-deoxyribose while RNA contains ribose. This leads to a greater resistance of DNA to alkaline hydrolysis, (2’-OH group in RNA makes the 3’-phosphodiester bond susceptible to nucleophilic cleavage) à it is selectively advantageous for the heritable form of genetic information to be DNA rather than RNA. 2. DNA contains thymine (T) instead of uracil (U). Cytosine deaminates to form uracil at ~ 100/day in a cell. Because C in one DNA strand pairs with G in the other strand, whereas U would pair with A, conversion of a C to a U results in a heritable mutation of a C-G pair to a U-A pair. To prevent this mutation a cellular repair mechanism (DNA glycosylases) “proofreads” DNA, and a U in DNA arising from C deamination is replaced by a C. If DNA normally contained U rather than T, this repair system could not readily distinguish U formed by C deamination from a natural U correctly paired with A. In fact, the T in DNA is a form of “U” (“T is 5-methyl-U”) à the 5-methyl group in T means “this form of U belongs to DNA à do not replace it.” OXIDATIVE AND CHEMICAL MUTAGENESIS IN DNA Oxidative damage Hydroxylation of guanine Mitochondrial DNA is most susceptible (presence of oxygen) Chemical alkylation Methylation of guanine Cells have mechanisms to correct most of these modifications (see DNA repair) OXIDATIVE AND CHEMICAL MUTAGENESIS IN DNA Example of G tautomers Promote deamination Lead to alkylation Cannot base pair with cytosine, but with thymine, causing a G:C to A:T transition in DNA MINOR NUCLEOSIDES IN DNA Modification is done after DNA synthesis 5-Methylcytidine is common in eukaryotes, but also found in bacteria N6-Methyladenosine is common in bacteria, not found in eukaryotes Epigenetic marker: Way to mark own DNA so that cells can degrade foreign DNA (prokaryotes) Way to mark which genes should be active (eukaryotes) Could the environment turn genes on and off in an inheritable manner? Minor bases of DNA (shown as nucleosides). 5Methylcytidine occurs in the DNA of animals and higher plants, N6-methyladenosine in bacterial DNA, and 5hydroxymethylcytidine in the DNA of bacteria infected with certain bacteriophages. MINOR NUCLEOSIDES IN RNA Inosine sometimes found in the “wobble position” of the anticodon in tRNA – Made by de-aminating adenosine – Provides richer genetic code Pseudouridine (Y ) found widely in tRNA and rRNA – More common in eukaryotes but found also in eubacteria – Made from uridine by enzymatic isomerization after RNA synthesis – May stabilize the structure of tRNA – May help in folding of rRNA Some minor bases of tRNAs (shown as the nucleosides). Inosine contains the base hypoxanthine. Note that pseudouridine, like uridine, contains uracil; they are distinct in the point of attachment to the ribose in uridine, uracil is attached through N-1, the usual attachment point for pyrimidines; in pseudouridine, through C-5. RADIATION-INDUCED MUTAGENESIS IN DNA UV light induces dimerization of pyrimidines; this may be the main mechanism for skin cancers. Ionizing radiation (X-rays and grays) causes ring opening and strand breaking. These are difficult to fix. Cells can repair some of these modifications, but others cause mutations. Accumulation of mutations is linked to aging and carcinogenesis. Formation of pyrimidine dimers induced by UV light. introduces a bend or kink into the DNA