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Biochemistry 3381A
Derek Mc Lachlin

Biochem 3381 Lecture 1 Phophosdiester bond joins nucleic acids together N-glycosidic bond joins base to backbone 3’ end can also be phosphorylated 10 bp per turn 1 turn is 34 angstroms (rise) 20 angstroms is the width of the helix Base tilt is 6 degrees A DNA – dehydrating conditions Wider and flatter 11.6 bp/turn Chi angles are anti Has a hole in the middle (no bases in the center of the helix) Z DNA – left handed and formed under high salt conditions Salt helps mask the charges so the phosphates can be closer together Must have alternating Py-Pu (CGCGCGCGCG) Chi angles are anti for Py and syn for Pu Major groove is shallower than minor groove Is real and it prevents the nucleosome core particles from forming Many bonds along the backbone (alpha, beta) but due to steric clashes, restricts bonds from rotating freely N-glycosidic bond (Chi) bond Py is always Anti, Pu can be either, if Syn = Z DNA Phosphate conformation BI = bases overlap, BII = bases more spaced out 3 Forces Stabilizing nucleic acids: Base stacking (vertical), Base pairing (horizontal), Ionic Interaction Happens in both DNA and RNA Base stacking result from van der Waals and partial charge interactions GC base pairs have higher base stacking energy than AT base pairs GC more stable, middle bond can’t be removed, anti-cooperativity Divalent cations especially Mg++ can stabilize ionic charges Absorbance changes upon denaturation (easy to follow) Factors influencing DNA stability: nature of the solvent, length and sequence of DNA strands, degree of complementarity, concentration and types of ion present, pH 15-25 degrees below Tm allows cooperative renaturing. Denaturing is also cooperative. RNA-RNA can’t form B-DNA extra OH steric clash so it looks like A-DNA RNA-DNA form an intermediate between A and B DNA Bask stacking can occur even for single stranded RNA RNA can form non-watson crick base pairs because of less backbone restrictions Lecture 2 3 main binding surfaces: major groove, minor groove, sugar-phosphate backbone Major groove – ATCG, Minor Groove - AT/CG, Sugar-phosphate backbone – DNA Helix turn Helix, most common motif Dimers bind to palindromic sequence Binds to both phosphate backbone and sequences Helix turn helix fits perfectly in B DNA Trp repressor H bonds mediated by water (all but one) Interactions are still specific H bonds are direct to phosphate backbone Indirect readout “flexibility of protein that allows for binding” Met repressor (homo dimeric) Beta strands are what interact with the protein DNA and protein adjust their conformation to make an induced fit (flexibility specific) One strand from each subunit fits in Binding of enzyme loosens the DNA for activity EcoRV: binding through a loop (unusual) still bending of DNA Zn++ ion coordinated by Cys (tetrahedral) and sometimes His Beta beta alpha structure Stable as independent unit Can be chained together, binds to 3 base pairs, long stretch is specific and non-specific Can have binuclear zinc fingers, dimer and bind far away Homeodomain ONLY IN EUKARYOTES Some crucial for domain Leucine zipper (coiled coil) Extended helixes interact with major groove, separated by half helix Basic region (bZIP) bHLH/Z combined motifs Nonspecific (histone, DNA repair), bind less tightly Prokaryotic: HTH and beta strands Eukaryotic: zinc fingers, homeodomains and leucine zippers Lecture 3 Supercoiling can happen in linear molecules when ends are contrained Twist – number of complete turns one strand makes around the axis of double helix (duplex axis) RH turns are (+), LH turns are (-) B DNA T=10bp/turn Underwound = decrease twist Overwound = increase twist Underwound RH DNA gives negative supercoiling (-) Overwound RH DNA gives positive supercoiling (+) Interwound writhe or plectonemic writhe Number of crossing over (RH negative, LH positive) Spiral or toroidal writhe Number of turns the duplex axis makes around the superhelix axis Negative spiral writhe is LH L=T+W In relaxed DNA, L = T because W = 0 For a closed circular double helix, L cannot be changed unless at least one strand is broken Change in T = 0 so change in L = change in W Type IA, relaxes negatively supercoiled DNA, increase writhe and linking number Found in every type of organism (increases L by 1) Increases writhe to 0, can’t make positive supercoiling Tyr bound to 5’ end Type 1B can relax negative or positive supercoiling Covalent bond between Tyr and 3’ end Not ATP driven, supercoil driven Type II topoisomerases Relax negatively or positively supercoiling Covalent bonds between Tyr and 5’end of each strand ATP required L changed in the steps of 2 (moving 2 strands) Gyrase, uses ATP to induce negative supercoilding (prokaryotic) Opposite to Type II topoisomerases (with ATP) Without ATP, works like Type II topoisomerase Transcription (Bacteria) rRNA (ribosomal) tRNA (delivery amino acids) mRNA (template for protein synthesis) snRNA (components of spliceosome) siRNA/miRNA (regulation of gene expression) snoRNAs (methylation of rRNAs) Gene: a polynucleotide that contains the information required to generate a polypeptide or RNA product and includes promoter and termination sequences (a part of gene) Structural gene: RNA or protein product that does something Bacterium has RNA Polymerase (5subunits) Synthesize all RNA except for primers (primase) Positively charged cleft (binds to negative DNA), looks like pincer Mg++ in the active site Core subunits: aaBB’w is sufficient for transcription but nonspecific Holoenzyme = aaBB’wO binds loosely to DNA in general but really tight to specific DNA Many O factors in bacteria (specificity) S70 is normal RNAP -10 region is the Pribnow box +1 is the initiation site Similarity of promoter to consensus affects rate of transcription More important genes have stronger promoter, less used gene has weaker promoter Different promoters have different consensus sequences Change temperature, different sigma factor Proteins act on both the sense strand and the anti-sense strand even though only sense is shown RNAP opens up 12 base pairs -10 -> +2 and does not required ATP and is reversible Hydrolysis of PPP is similar to hydrolysis of ATP Abortive initiation RNAP is too tight and does not dislodge so it leaves After RNAP escapes the promoter, the sigma factor is not required nor is clamp protein 20-50nt/sec but structure can cause it to stall or stop RNAP has error rate in 1 in 10000 RNAP rotates relative to helix DNA ahead of transcription bubble becomes positively supercoiled (topoisomerase I) DNA behind of transcription bubble becomes more negatively supercoiled (gyrase) Corrected by topoisomerases and DNA gyrase After one RNAP has escaped, more can attach and begin transcription Termination 1 type: mediated by intrinsic terminator region – palindromic GC rich region followed by 4-10 A’s AU pairing is weak promoting dissociation Stability of hairpin is important for intrinsic termination nd 2 type: Rho factor, a hemohexameric helicase Each unit has 2 domain, RNA binding domain at N terminal, ATPase in C domain Binds C-rich regions but sequence is not well defined, but no hairpins Normally open, when strand comes, makes closed conformation and ATP pulls tail down Rho is always bound, when a sequence it likes comes along, it will go into Rho When RNA is short, RNAP has an allosteric change Transcription (Eukaryotic) Multiple eukaryotic RNAPs mRNAs are all monocistronic More complex machinery More complicated promoter/control/regulation/expression levels RNAPI in nucleoli rRNA RNAPII in nucleoplasm mRNAs - MAJOR RNAPIII in nucleoplasm 5S rRNA, tRNAs and other RNAs Mitochondral/Chloroplast RNA for their genes Yeast RNAP II RPB1/RPB2 are homologous B and B’ subunits in RNAP Mg++ in active site Zinc ions as well, all over the place C-terminal domain in RPB1 subunit Tandem repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser Ser can be phosphorylated Two main types of eukaryotic promoter Sharp: start sites are clearly defined, usually have “core promoter” elements, TATA box, Inr Broad: not well defined, still within that area but no specific nucleotide “CpG island” RNAPII core promoters are specific and only in some genes (tissue specific) 1/3 have TATA and ½ have Inr Different elements combine to form the promoter region (in contrast, all bacteria have -10) Some promoters are after the transcription site (get transcribed) TATA box and Initiator CpG islands, especially those that are constitutively expressed Cytosine base is usually methylated in the CpG islands Degree of methylation correlates with the amount of expression (methylate is shut down) When you methylate C, it looks like Thymine In deamination, C becomes U and is fixed easily but when C->T, cell has no idea how to fix Some CpG islands are in the middle of the gene (don’t know why) General transcription factors are similar to sigma in bacteria Unlike sigma (one protein), TFs can be very big proteins Two models: sequential binding of individual factors or binding of pre-formed complex Both exist in vivo, happens both ways Minimal PIC: TATA Binding Protein (TBP, TFIIB, TFIIE, TFIIF, TFIIH) TFIIA and TFIID is not necessary, but may make it better TFIIA stabilizes TBP TFIIB orients TBP TFIIF recruits RNAPII TFIIE recruits TFIIH (helicase) which induces open complex TATA binding protein (TBP) does not need TATA but it knows where to bind Kinks and partially unwinds DNA Also required for transcription by RNAP I and III Monomer but looks like dimer (needs orientation) RNAPII is unphosphoylated when recruited TFIIH kinase activity phosphorylates CTD of RPB1 of RNAPII on Ser5 of repeat, add negative charges, causes tail to adopt an extended conformation Signals for the start of transcription (TFIIH last to be recruited) When phosphorylated, allows elongation factors, RNA processing enzymes, and histone-modifying proteins bind to CTD (350bps) TFIIH and TFIIF remain with RNAP II while other GTFs dissociate Phosphorylation breaks off the GTFs RPB2 and RPB1 make a claw Improper Watson crick base pairs simply diffuse away Rudder separates RNA from template DNA Bridge may bend to move template through enzyme Proofre
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