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Replication, Transcription, and RNA Processing

Replication, Transcription, and RNA Processing. Andy Howard Biochemistry Lectures, Spring 2019 Thursday 7 March 2019. Nucleic Acids and Central Dogma. We’ll finish our description of types and functions of RNA

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Replication, Transcription, and RNA Processing

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  1. Replication, Transcription, and RNA Processing Andy HowardBiochemistry Lectures, Spring 2019Thursday 7 March 2019

  2. Nucleic Acids and Central Dogma • We’ll finish our description of types and functions of RNA • We’ll go through the Central Dogma concepts of replication, transcription and translation, along with some of the post-transcriptional modifications that happen to RNA RNA and Central Dogma

  3. RNA Types Functions DNA replication Semi-conservative Prokaryotic Eukaryotic Repair Recombination Transcription RNA polymerase Steps required RNA Processing What we’ll cover RNA and Central Dogma

  4. tRNA structure: overview RNA and Central Dogma

  5. Amino acid linkage to acceptor stem Amino acids are linked to the 3'-OH end of tRNA molecules by an ester bond formed between the carboxyl group of the amino acid and the 3'-OH of the terminal ribose of the tRNA. RNA and Central Dogma

  6. Ribosomal RNA • rRNA: catalyic and scaffolding functions within the ribosome • Responsible for ligation of new amino acid (carried by tRNA) onto growing protein chain Haloarcula marismortui 23S rRNA602 bases PDB 1FFZ, 3.2Å RNA and Central Dogma

  7. Ribosomal RNA, continued Can be large: mostly 500-3000 bases a few are smaller (150 bases) Very abundant: 80% of cellular RNA Relatively slow turnover RNA and Central Dogma

  8. Small RNA • sRNA: few bases / molecule • often found in nucleus; thus it’s often called small nuclear RNA, snRNA • Involved in various functions, including processing of mRNA in the spliceosome Human Protein Prp31complexed to U4 snRNA33 bases + 85kDa heterotetramerPDB 2OZB, 2.6Å RNA and Central Dogma

  9. Small RNAs, continued Some are catalytic Typically 20-1000 bases Not terribly plentiful: ~2 % of total RNA RNA and Central Dogma

  10. Other small RNAs snoRNA77courtesy Wikipedia • 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 RNA and Central Dogma

  11. siRNAs &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 Viral p19 protein complexed to human 19-base siRNA17kDa protein PDB 1R9F, 1.95Å RNA and Central Dogma

  12. Significance of RNAi This is a form of gene silencing or RNA interference RNAi also changes chromatin structure and has long-range influences on expression RNA and Central Dogma

  13. Unusual bases in RNA • mRNA, many sRNAs are mostly ACGU • rRNA, tRNA, some sRNAs have more odd ones Often modified in place within the (t)RNA molecule RNA and Central Dogma

  14. iClicker quiz question 1 1. Which of these sequences is palindromic? • (a) C-A-T-G-G-T-A-C • (b) C-A-T-A-T-G • (c) C-G-C-G-C-G-C • (d) all of the above • (e) none of the above RNA and Central Dogma

  15. iClicker quiz question 2 2. An RNA molecule consists of 81 bases, including 10 nonstandard bases. It is probably • (a) mRNA • (b) tRNA • (c) rRNA • (d) sRNA • (e) not real RNA at all RNA and Central Dogma

  16. Central Dogma • The Central Dogma describes replication, transcription, and translation as the core events of molecular biology • There are subtleties in between, but that three-step process is still significant. • Thus here we complete a discussion of replication, transcription, and then move on to RNA processing and translation. RNA and Central Dogma

  17. Semi-conservative replication Photo courtesy U. Costa Rica • A bit of a fanciful term; refers to the fact that, during DNA replication, each daughter molecule contains one of the strands of the parent • Each daughter contains 1/2 (semi) of original molecule • This mode of inheritance was predicted by the Watson/Crick model RNA and Central Dogma

  18. 3 models RNA and Central Dogma

  19. Meselson & Stahl • 1958: showed that DNA really is replicated this way • DNA grown with 15N has higher density • 15N DNA allowed to replicate exactly once has intermediate density RNA and Central Dogma

  20. Meselson-Stahlexperiment • Note that the bottom 2 density gradients are for mixtures of generations RNA and Central Dogma

  21. The E.coli chromosome • One circular, double-stranded DNA molecule of about 4.6*106bp • Replication begins in only one place, i.e. a single origin of replication (OriC in E.coli) • Replication moves both directions until the two replication efforts meet at the termination site RNA and Central Dogma

  22. E.coli replisome Protein machine that accomplishes replication is the replisome;one replisome in each direction Replication forks move 1000 bp/sec; thus E.coli can be replicated in 38 min (2280 sec) RNA and Central Dogma

  23. How replication works in prokaryotes • Takes place in the cytosol: there is no nucleus • Specific enzymes form the molecular machine to carry out the task • Has to involve separation of the strands • Process divided into initiation, elongation, and termination • Enzymatic functions identified for each segment RNA and Central Dogma

  24. Prokaryotic DNA polymerases • Several varieties • DNA polymerase III is the one responsible for most of the work (but the 3rd discovered);it’s the biggest and most complex • DNA pol I involved in error correction and helps with replication of one of the strands • DNA pol II also does DNA repair • Multi-subunit, complex entities RNA and Central Dogma

  25. DNA Pol III Diagram from Kelman et al(1998) EMBO J. 17:2436 RNA and Central Dogma

  26. Components of DNA Pol III RNA and Central Dogma

  27. So how does it work? • Add 1 nucleotide @ a time to 3’ end of growing chain • Substrate is a dNTP • Watson-Crick bp determines specificity • Enzyme spends 75% of time tossing out wrong bases • Forms phosphodiester linkage • Pol III remains bound to the replication fork Diagram from answers.com RNA and Central Dogma

  28. Error correction in DNA pol III • 3’-5’ proofreading recognizes incorrectly paired bases and repairs most of them • This is an exonuclease activity because it clips off the last nucleotide in the chain • 10-5 inherent error rate drops to 10-7 because the exonuclease goofs 1% of the time • Separate repair enzymes drop that down to 10-9 RNA and Central Dogma

  29. Processivity • Refers to fact that many nucleotides can be added to a growing chain following a single association event in which the polymerase (e.g. E.coli Pol III) associates with the template DNA. • We describe replication as highly processive if 50,000 bases can be replicated based on a single association of Pol III with our template. •  subunits slide along, which is how this is done •  complex is responsible for keeping the polymerase attached so that this is possible RNA and Central Dogma

  30. Leading & lagging strands 3’ Fork movement 5’ Leading strand Parental strand 5’ 3’ 5’ Parental strand Lagging strand 3’ 3’ 5’ Replication on the leading strand is straightforward because it’s moving the same direction as the fork. Replication on the lagging strand is discontinuous and somewhat more complex RNA and Central Dogma

  31. Leading & lagging strands: dynamics • Leading strand • Begins at the origin • Ends at termination site • Continuous polynucleotide • Lagging strand • Built in short pieces, opposite to fork movement • Each Okazaki fragment starts with an RNA primer made in the primosome • Fragments joined via DNA Pol I and DNA ligase RNA and Central Dogma

  32. Leading-strand synthesis • One base at a time is incorporated by a subunit of DNA polymerase, complementary to existing strand • At some point RNA primer is replaced with DNA Image courtesy U.Pittsburgh RNA and Central Dogma

  33. Lagging-strand synthesis • Movement of enzyme is opposite to unwinding • It must work a few bases (~1000) at a time and then back up • Segments thus formed on the lagging strand are known as Okazaki fragments • DNA Pol I removes RNA primer • DNA ligases link together Okazaki fragments RNA and Central Dogma

  34. Primases • These are DNA-dependent RNA polymerase enzymes that initiate DNA synthesis, particularly on the lagging strand, where you need to do that at the beginning of each Okazaki fragment Bacillus stearothermophilus DnaG helicase binding domain347 kDa trimer ofheterotrimersPDB 2R6A, 2.9Å RNA and Central Dogma

  35. The replisome • Molecular machine responsiblefor 2-strand DNA synthesis:Primosome, DNA Pol III,other proteins • Requires SS-DNA coated with SSB so a helicase (part of primosome) unwinds the DNA and the SSB keeps it from folding back during replication RNA and Central Dogma

  36. Initiation • Begins in E.coli at a single origin called OriC • DnaA binds to origin—region called DnaA box • Replication fork forms after it binds • Helicases & primasesset up for starting replication • Complementary RNA tag attached at replication fork RNA and Central Dogma

  37. Elongation • DNA polymerase operates in 5’-3’ directionon both strands • For leading strand that’s straightforward:replication moves in direction of unwinding of DNA • For lagging strand it’s more complex, since it’s moving the wrong way RNA and Central Dogma

  38. Termination • Replication needs to know how to stop; prevents the replication forks from passing through the site • Defined sequence ter is opposite the origin on the chromosome • Specific enzyme, Tus, involved in recognizing termination signals • Ter has sequences that play a role in separating the daughter chromosomes Tus-Ter complex;images courtesy Memorial Univ., Newfoundland RNA and Central Dogma

  39. Role of DNA Pol I • Part of the system for producing a continuous DNA strand on the lagging side • Contains both 5’3’ polymerase activity and 3’5’ proofreading exonuclease activity • Also has 5’3’ exonuclease activity: that’s used to remove the RNA primer RNA and Central Dogma

  40. Rates & sequencing • Because there’s only one place where replication can begin, the process must occur in discrete steps • The enzymes themselves are efficient, because they move with the unwinding of the double helix • Typical rates 1000 nucleotides/ sec • So for E.coli it takes 38 min = 2280 sec to replicate the entire chromosome: (4.6*106 bp) /[(103 bp/sec)(2 directions)] = 2300 sec RNA and Central Dogma

  41. Where are things happening? • Both leading- and lagging-strand synthesis are catalyzed in both the clockwise and counterclockwise directions. • Each DNA Pol III molecule is catalyzing both leading- and lagging-strand synthesis. RNA and Central Dogma

  42. Eukaryotes • Bigger chromosomes, more of them • Chromosomes are rarely circular • Fruit-fly chromosomes: • Sex chromosome, 2 long autosomes,one tiny autosome • 1.65 * 108bp, 14000 genes • ~6000 replication forks, i.e. 3000 origins Drosophila chromosomeTEM reconstruction RNA and Central Dogma

  43. Eukaryotic replication, continued • Human • 22 pairs of autosomes, sex chromosome • 3.4*109bp, ~22000 genes • Replication is bidirectional as in E.coli • More than one origin so replication is comparably fast even though rate is lower • Origins in active regions of genome get replicated early in S; slower ones later in S RNA and Central Dogma

  44. Eukaryotic polymerases Human mitochondrial DNA pol γ-2EC 2.7.7.7104 kDa dimerPDB 3IKL, 3.10Å >= 5 different ones:most important nuclearpolymerases are δ, ε γ is mitochondrial,α does primers, repair Okazaki fragments handled as with prokaryotes PCNA acts like the E.coli β subunit RNA and Central Dogma

  45. DNA replication: accuracy! • The extraordinary fidelity of heritance in prokaryotes and eukaryotes derives from the net accuracy of DNA replication. We’ll outline the steps of replication and the proofreading that goes with it. RNA and Central Dogma

  46. DNA repair • DNA is the only macromolecule that gets repaired: it’s too important not to • A single base error can be fatal, even in prokaryotes • Natural rates of misincorporation are small but nonzero • Rate can go up upon exposure to ionizing radiation, some chemicals, some toxins RNA and Central Dogma

  47. Direct repair • Enzymes scan DNA for particular lesions • Pyrimidine dimers are noted and repaired this way • Some can replace the base without breaking the phosphodiester backbone Image courtesy U. München RNA and Central Dogma

  48. Excision repair • Endonuclease recognizes lesion • Cleaves upstream &downstream —12-13 bases • Only cleaves damaged strand • Removal may require helicase • DNA polymerase (I in prokaryotes) fills the gap • DNA ligase reseals the lesion Diagram courtesy Beth Montelone, Kansas State U. RNA and Central Dogma

  49. Other repairs H2O • Repairing hydrolytic deamination of A, C, G: • DNA glycosylase flips base out and hydrolyzes glycosidic bond • Endonuclease sutures in one replacement base (sometimes part of same protein) NH3 RNA and Central Dogma

  50. Recombination • Recombination is any exchange or transfer of DNA from one spot to another • Homologous recombination involves exchanges in closely-related sequences; can involve paired chromosomes • Transposons are elements that can be readily recombined nonhomologously RNA and Central Dogma

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