Nucleic acid metabolism

1. Nucleic acid metabolism Andy HowardBiochemistry Lectures, Fall 201029 November 2010 Nucleic Acid Metabolism

2. Nucleic Acids • We’ll complete our discussion of RNA and polynucleotide cleavage • Then we’ll look at biosynthesis of purine nucleosides Nucleic Acid Metabolism

3. RNA Differences Hydrolysis of Nucleotides RNA and DNA Restriction enzymes PRPP Pyrimidine Synthesis Path to Uridylate Cytidylate What we’ll cover Nucleic Acid Metabolism

4. RNA physics & chemistry • RNA molecules vary widely in size, from a few bases in length up to 10000s of bases • There are several types of RNA found in cells Type % %turn- Size, Partly Role RNA over bases DS? mRNA 3 25 50-104 no protein template tRNA 15 21 55-90 yes aa activation rRNA 80 50 102-104 varies transl. catalysis & scaffolding sRNA 2 4 30-103 varies various Nucleic Acid Metabolism

5. Unusual bases in RNA • mRNA, sRNA mostly ACGU • rRNA, tRNA have some odd ones Nucleic Acid Metabolism

6. Small RNAs • 21-28 nucleotides • Target RNA or DNA through complementary base-pairing • Several types, based on function: • Small interfering RNAs (q.v.) • microRNA: control developmental timing • Small nucleolar RNA: catalysts that (among other things) create the oddball bases snoRNA77courtesy Wikipedia Nucleic Acid Metabolism

7. siRNAs and gene silencing • Small interfering RNAs block specific protein production by base-pairing to complementary seqs of mRNA to form dsRNA • DS regions get degraded & removed • This is a form of gene silencing or RNA interference • RNAi also changes chromatin structure and has long-range influences on expression Viral p19 protein complexed to human 19-base siRNA PDB 1R9F1.95Å 17kDa protein Nucleic Acid Metabolism

8. Do the differences between RNA and DNA matter? Yes! • DNA has deoxythymidine, RNA has uridine: • cytidine spontaneously degrades to uridine • dC spontaneously degrades to dU • The only dU found in DNA is there because of degradation: dT goes with dA • So when a cell finds dU in its DNA, it knows it should replace it with dC or else synthesize dG opposite the dU instead of dA Nucleic Acid Metabolism

9. Ribose vs. deoxyribose • Presence of -OH on 2’ position makes the 3’ position in RNA more susceptible to nonenzymatic cleavage than the 3’ in DNA • The ribose vs. deoxyribose distinction also influences enzymatic degradation of nucleic acids • I can carry DNA in my shirt pocket, but not RNA Nucleic Acid Metabolism

10. Backbone hydrolysis of nucleic acids in base • Nonenzymatic hydrolysis in base occurs with RNA but not DNA, as just mentioned • Reason: in base, RNA can form a specific 5-membered cyclic structure involving both 3’ and 2’ oxygens • When this reopens, the backbone is cleaved and you’re left with a mixture of 2’- and 3’-NMPs Nucleic Acid Metabolism

11. Enzymatic cleavage of oligo- and polynucleotides • Enzymes are phosphodiesterases • Could happen on either side of the P • 3’ cleavage is a-site; 5’ is b-site. • Endonucleases cleave somewhere on the interior of an oligo- or polynucleotide • Exonucleases cleave off the terminal nucleotide Nucleic Acid Metabolism

12. An a-specific exonuclease Nucleic Acid Metabolism

13. A b-specific exonuclease Nucleic Acid Metabolism

14. Specificity in nucleases • Some cleave only RNA, others only DNA, some both • Often a preference for a specific base or even a particular 4-8 nucleotide sequence (restriction endonucleases) • These can be used as lab tools, but they evolved for internal reasons Nucleic Acid Metabolism

15. Variety of nucleases Nucleic Acid Metabolism

16. Restriction endonucleases • Evolve in bacteria as antiviral tools • “Restriction” because they restrict the incorporation of foreign DNA into the bacterial chromosome • Recognize and bind to specific palindromic DNA sequences and cleave them • Self-cleavage avoided by methylation • Types I, II, III: II is most important • I and III have inherent methylase activity; II has methylase activity in an attendant enzyme Nucleic Acid Metabolism

17. What do we mean by palindromic? • In ordinary language, it means a phrase that reads the same forward and back: • Madam, I’m Adam. (Genesis 3:20) • Eve, man, am Eve. • Sex at noon taxes. • Able was I ere I saw Elba. (Napoleon) • A man, a plan, a canal: Panama! (T. Roosevelt) • With DNA it means the double-stranded sequence is identical on both strands Nucleic Acid Metabolism

18. Quirky math question to ponder • Numbers can be palindromic:484, 1331, 727, 595… • Some numbers that are palindromic have squares that are palindromic…222 = 484, 1212 = 14641, . . . • Question: if a number is perfect square and a palindrome, is its square root a palindrome? (answer will be given orally) Nucleic Acid Metabolism

19. Palindromic DNA • G-A-A-T-T-C • Single strand isn’t symmetric: but the combination with the complementary strand is: • G-A-A-T-T-CC-T-T-A-A-G • These kinds of sequences are the recognition sites for restriction endonucleases. This particular hexanucleotide is the recognition sequence for EcoRI. Nucleic Acid Metabolism

20. Cleavage by restriction endonucleases • Breaks can be • cohesive (if they’re off-center within the sequence) or • non-cohesive (blunt) (if they’re at the center) • EcoRI leaves staggered 5’-termini: cleaves between initial G and A • PstI cleaves CTGCAG between A and G, so it leaves staggered 3’-termini • BalI cleaves TGGCCA in the middle: blunt! Nucleic Acid Metabolism

21. iClicker question 3: • 3. Which of the following is a potential restriction site? • (a) ACTTCA • (b) AGCGCT • (c) TGGCCT • (d) AACCGG • (e) none of the above. Nucleic Acid Metabolism

22. Example for EcoRI • 5’-N-N-N-N-G-A-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-A-G-N-N-N-N-5’ • Cleaves G-A on top, A-G on bottom: • 5’-N-N-N-N-GA-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-AG-N-N-N-N-5’ • Protruding 5’ ends:5’-N-N-N-N-GA-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-AG-N-N-N-N-5’ Nucleic Acid Metabolism

23. How often? • 4 types of bases • So a recognition site that is 4 bases long will occur once every 44 = 256 bases on either strand, on average • 6-base site: every 46= 4096 bases, which is roughly one gene’s worth Nucleic Acid Metabolism

24. EcoRI structure • Dimeric structure enables recognition of palindromic sequence •  sandwich in each monomer EcoRI pre-recognition complex PDB 1CL8EC 3.1.21.4, 1.8Å 57 kDa dimer + DNA Nucleic Acid Metabolism

25. Methylases HhaI methyltransferasePDB 1SVU2.66Å; 72 kDa dimerEC 2.1.1.37, 2.66Å • A typical bacterium protects its own DNA against cleavage by its restriction endonucleases by methylating a base in the restriction site • Methylating agent is generally S-adenosylmethionine Structure courtesy steve.gb.com Nucleic Acid Metabolism

26. Use of restriction enzymes • Nature made these to protect bacteria; we use them to cleave DNA in analyzable ways • Similar to proteolytic digestion of proteins • Having a variety of nucleases means we can get fragments in multiple ways • We can amplify our DNA first • Can also be used in synthesis of inserts that we can incorporate into plasmids that enable us to make appropriate DNA molecules in bacteria Nucleic Acid Metabolism

27. Phosphoribosyl pyrophosphate PRPP synthetase • Activation of ribose-5-P (see Calvin cycle, etc.) by ATP:-ribose-5-P + ATP  PRPP + AMP • Has roles in other systems too PRPP synthetasePDB 2H06 215 kDa hexamerdimer shown; human EC 2.7.6.1, 2.2Å Nucleic Acid Metabolism

28. Carbamoyl phosphate Pyrimidine synthesis:carbamoyl aspartate • Uridine is based on orotate,which is derivated fromcarbamoyl aspartate • We’ve already seen the carbamoyl phosphate synthesis back in chapter 17 via carbamoyl phosphate synthetase • Carbamoyl phosphate + aspartate carbamoyl aspartate + Pivia aspartate transcarbamoylase Carbamoyl aspartate Nucleic Acid Metabolism

29. Aspartate transcarbamoylase • ATCase is the classic allosteric enzyme • E.coli version is inhibited by pyrimidine nucleotides and activated by ATP • CTP by itself is 50% inhibitory; CTP+ UTP is almost totally inhibitory ATCasePDB 1D09Trimer of heterotetramers1 heterotetramer shown (cf. fig.18.11)EC 2.1.3.2, 2.1Å E.coli Nucleic Acid Metabolism

30. Carbamoyl aspartate to dihydroorotate • Carbamoyl aspartate dehydrates and cyclizes to L-dihydroorotate via dihydroorotase • TIM barrel protein Dihydro-orotate PDB 2Z2678 kDa dimerEC 3.5.2.3, 1.3ÅE.coli Nucleic Acid Metabolism

31. Dihydroorotate to orotate • Ubiquinone acts as oxidizing agent reducing the 5 & 6 Carbons via dihydroorotate dehydrogenase • Some versions incorporate FMN PDB 2E6F69 kDa dimerEC 1.3.3.11.26ÅTrypanosoma cruzi Nucleic Acid Metabolism

32. Adding phosphoribose • Orotate + PRPP  orotidine 5’-monophosphate + PPi • Usual argument re pyrophosphate hydrolysis • Enzyme: orotidine phosphoribosyl transferase PDB 2PS151 kDa dimerYeastEC 2.4.2.10, 1.75Å Orotidine 5’-monophosphate Nucleic Acid Metabolism

33. Decarboxylation • OMP decarboxylated to form UMP via OMP decarboxylase • Bacterial forms are TIM barrel proteins • Acceleration is 1017-fold relative to uncatalyzed rate PDB 1KLY54 kDa dimerEC 2.4.2.10, 1.75ÅMethanobacteriumthermo-autotrophicum Nucleic Acid Metabolism

34. Eukaryotic variation • Orotate produced in the mitochondrion moves to the cytosol • UMP synthase combines the last two reactions—orotidine to OMP to UMP OMP decarboxylasedomainof UMP synthase PDB 2P1F64 kDa dimerhuman1.76Å Nucleic Acid Metabolism

35. UMP to UTP • Uridylate kinase converts UMP to UDP:UMP + ATP  UDP + ADPenzyme is related to several amino acid kinases • Nucleoside diphosphate kinase exchanges di for tri:UDP + ATP  UTP +ADP(non-specific enzyme) Uridylate kinase PDB 1UKZ24 kDa monomerSaccharomycesEC 2.7.4.-, 1.9Å Nucleic Acid Metabolism

36. CTP synthetase • UTP + gln + ATP CTP + glu + ADP + Pi • Glutamine side-chain is amine donor • ATP provides energy •  sandwich (Rossmann) • Enzyme is inhibited by CTP • In E.coli, it’s activated by GTP (makes sense!) PDB 1S1M247 kDa tetramerdimer shown EC 6.3.4.2, 2.3ÅE.coli Nucleic Acid Metabolism

37. Purine synthesis • Considerably more complex than pyrimidine synthesis • More atoms to condense and two rings to make • More ATP to sacrifice during synthesis • Several synthetase (ligase) reactions require ATP • Based on PRPP, gln, 10-formyl THF, asp Nucleic Acid Metabolism