1. Replication, Transcription, and RNA Processing
Andy Howard Biochemistry Lectures, Spring 2019 Thursday 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
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RNA and Central Dogma

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

4. tRNA structure: overview
RNA and Central Dogma
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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.
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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 rRNA 602 bases
PDB 1FFZ, 3.2Å
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7. Ribosomal RNA, continued
Can be large: mostly bases
a few are smaller (150 bases)
Very abundant: 80% of cellular RNA
Relatively slow turnover
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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 Prp31 complexed to U4 snRNA 33 bases + 85kDa heterotetramer PDB 2OZB, 2.6Å
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9. Small RNAs, continued Some are catalytic Typically 20-1000 bases
Not terribly plentiful: ~2 % of total RNA
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10. Other small RNAs 21-28 nucleotides
snoRNA77 courtesy 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
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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 siRNA 17kDa protein
PDB 1R9F, 1.95Å
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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
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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
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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
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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
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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.
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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
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18. 3 models
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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
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20. Meselson-Stahl experiment
Note that the bottom 2 density gradients are for mixtures of generations
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21. The E.coli chromosome
One circular, double-stranded DNA molecule of about 4.6*106 bp
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
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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)
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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
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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
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25. DNA Pol III Diagram from Kelman et al (1998) EMBO J. 17:2436 3/7/2019
RNA and Central Dogma

26. Components of DNA Pol III
Subunit
Mr,kDa
Gene
Activity a
130
polC/dnaE
Polymerase e 27
dnaQ/mutD
3’-5’ exonuclease q
8.9
holE
Stabilizes proofreading by ? b 40
dnaN
Sliding clamp t 71
dnaX
Dimer, ATPase
g complex
15-47
Various
Processivity
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27. So how does it work?
Add 1 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
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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
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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
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30. Leading & lagging strands3’
Fork movement 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
Leading strand
Parental strand 5’ 3’ 5’ 3’
Parental strand
Lagging strand 3’ 5’
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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
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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
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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
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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 domain 347 kDa trimer of heterotrimers PDB 2R6A, 2.9Å
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35. The replisome
Molecular machine responsible for 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
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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 & primases set up for starting replication
Complementary RNA tag attached at replication fork
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37. Elongation DNA polymerase operates in 5’-3’ direction on 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
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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
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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
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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
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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.
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42. Eukaryotes Bigger chromosomes, more of them
Chromosomes are rarely circular
Fruit-fly chromosomes:
Sex chromosome, 2 long autosomes, one tiny autosome
1.65 * 108 bp, genes
~6000 replication forks, i.e origins
Drosophila chromosome TEM reconstruction
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43. Eukaryotic replication, continued
Human
22 pairs of autosomes, sex chromosome
3.4*109 bp, ~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
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44. Eukaryotic polymerases
>= 5 different ones: most important nuclear polymerases are δ, ε
γ is mitochondrial, α does primers, repair
Okazaki fragments handled as with prokaryotes
PCNA acts like the E.coli β subunit
Human mitochondrial DNA pol γ-2 EC kDa dimer PDB 3IKL, 3.10Å
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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.
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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
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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
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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.
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49. Other repairs 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)
H2O
NH3
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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
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51. Recombination as repair
Bad lesions are simply skipped
Intact strand from one daughter acts as template for repairing broken strand
Most recombination genes play roles in repair too
E.coli RecA in compressed helical form
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52. Repair & Disease
Repair deficiencies render the organism susceptible to mutation-related maladies
BRCA1&BRCA2 are proteins that bind to RecA in humans and help repair DSBs
So some mutations in these proteins leave people prone to cancer
BRCA1 BRCT domains complexed to BACH1 helicase
PDB 1T15, 1.85Å
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53. iClicker question #3
3. Why should the beta subunit in bacterial polymerases function as a sliding clamp?
(a) to isolate the currently processed base so that it doesn’t get deleted
(b) to speed the replication process
(c) to reduce error rates
(d) to prevent creation of Holliday junctions
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54. Transcription: overview
Roger Kornberg
Transcription: overview
Transcription is the process of creating an RNA molecule based on a DNA template
Mechanism applies to all forms of RNA, not just mRNA
Accomplished through a molecular machine called RNA polymerase, using NTPs as substrates
Taq RNA polymerase during elongation
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55. Elongation Resembles elongation during DNA replication
Basic reaction: RNAn–OH + NTP RNAn+1–OH + PPi
Replication vs. transcription elongation:
Property
Replication
Transcription
Substrate
dNTP
NTP
Interaction with template
Long distance
Shorter distance
Product
dsDNA
ssRNA
Rate
~1000 nuc/sec
30-85 nuc/sec
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56. RNA polymerase structure
Stoichiometry α2ββ’ω
β, β’ form a groove at one end: DNA binds, polymerization occurs there
Groove big enough to hold 16bps
2 α subunits provide assembly scaffold: other subunits, other proteins
σ not part of of core polymerase α2
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57. Error rates during elongation
Typically around 10-6
That’s worse than replication because there is no built-in proofreading
Accuracy less critical than with DNA, since RNA doesn’t get passed on!
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58. Transcription initiation
Occurs at sites called promoters
Bacterial systems: several genes activated by a single promoter
Eukaryotic systems: generally one promoter region per gene
We distinguish between the DNA’s coding strand (the one that looks like the transcript) and the template strand (the one that’s actually being transcribed)
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59. How the process begins
≥1 proteins bind to promoter & to the RNA polymerase
These proteins direct the polymerase to the promoter site
Bacterial: σ subunit of RNA pol does this
Promoter regions are not identical but they contain commonalities: consensus sequences
Region 4 of E.coli σ subunit + DNA
16 kDa protein dimer PDB 1KU7, 2.4Å
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60. Typical sequences Binding to 2 regions
σ70 TATA box sequence: TATAAT
Typical sequences
Binding to 2 regions
10 bp upstream (the TATA box)
35 bp upstream (-35 region)
How strong the promoter is (i.e., how efficient transcription is) is correlated with how close the homology to the consensus sequence is
Different sigma subunits have different consensus sequences
σ region: TTGACA
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61. What do sigma subunits do?
Core polymerase binds DNA indiscriminantly, dissociates slowly: Ka ~ 1010M-1, t½~1 hr
Holoenzyme:
binds promoter with Ka=2*1011M-1, t½=2.5h
binds other regions Ka=5*106M-1, t½=3 sec
Quick because of one-dimensional diffusion of RNA polymerase along the DNA:
Holoenzyme can scan 2000 bp looking for promoters
Restriction enzymes do the same thing
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62. How it starts
RNA pol holoenzyme can start polynucleotide synthesis on its own
Unwinding: polymerase+promoter undergo conformational change: RPc  RPo with 18bp unwound
Template strand positioned
2 NTPs move in, H-bonded to template strand nucleotides, phosphodiester linkage formed
~ 8 more bases added; then enzyme switches from initiation to elongation (conf change), clears promoter, sigma falls off, others bind
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63. Disassembly: two mechanisms
Elongation complex is unstable: transcription complex falls off by itself
More likely in pause sites, e.g. GC-rich regions
Even more likely in palindromic regions
Protein rho helps disassembly
Hexameric ATPase, high affinity for ssRNA
May be an RNA-DNA helicase
Binds to ssRNA exposed behind paused complex; ATP hydrolyzed, RNA wraps around rho, makes the transcript fall off
Coupled to translation termination in bacteria
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64. Transcription in eukaryotes
3 nuclear core RNA polymerases, also mitochondrial and chloroplast polymerases
pol I: big rRNA; found in nucleolus
pol II: pre-mRNAs, snRNAs (U1,U2,U4, U5)
pol III: other sRNAs, tRNA, small rRNAs
Promoter complexes (notably for pol II) require transcription factors TFIIB,D,E,F,H, others
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65. Exemplary interaction: TBP bound to TATA-box
TBP part of TFIID:
Beta sheet lies in minor groove
Promoter DNA is bent
Same subunit used to initiate transcription with RNA pol I and III
Arabidopsis TBP + TATAAAAG 62 kDa complex PDB 1QNA, 1.8Å
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66. Chromatin’s role
A huge percentage of genomic DNA remains untranscribed within any given cell
Most of the decision-making occurs at the level of the chromatin complexes in the nucleosome
Histones: acetylations, deacetylations, methylations of K; methylations of R; phosphorylations of S & T are signals
Nucleosome positioning & remodeling
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67. How is chromatin controlled?
Protein complexes use ATP hydrolysis to remodel the nucleosomes and provide access of proteins to the DNA
Some of the complexes contain HATs or HDACs
J.Lindsay et al. (2005) Neurosurg. Focus 19:5
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68. Regulation of transcription
A few genes are transcribed richly in every cell: they have strong promoters
Many mechanisms for regulating the rest
Both repressors and activators exist
Typically allosteric proteins whose functions are influenced by ligand binding
For genes that code for metabolic enzymes, the cell can control transcription to make available the appropriate proteins only when it’s useful
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69. Repression examples
Example 1: repressor for metabolic genes prevents transcription in absence of substrate; when substrate (“inducer”) binds to repressor, it stops repression and allows transcription; this means no needless transcription
Example 2: repressor operates only when downstream metabolite (“corepressor”) binds to repressor; this prevents overproduction on pathway
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70. β-galactosidase processing
E.coli doesn’t ordinarily transcribe the genes required for lactose processing in high quantities
If it needs to rely on β-galactosides, it will
Three proteins required:
Protein
Gene
Function
Lactose permease
lacY
Symport transporter
β-galactosidase
lacZ
Hydrolysis to monosaccharides
Thiogalactoside transacetylase
lacA
Acetylation helps eliminate toxic products
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71. The lac operon
These 3 genes: transcribed from single promoter to produce one big mRNA
Homotetrameric regulatory protein called lac repressor coded for by lacI
Repressor binds to two “operator” sites near promoter: O1 next to promoter, O2 within coding region of lacZ
E.coli lac repressor
155 kDa tetramer
PDB 3EDC, 2.1Å
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72. Kinetics of repression
Ka(O1) = 1013M-1
Kinetics of repression
When repressor binds to both operators, it pushes the DNA into a stable loop
Either prevents binding of polymerase or prevents conversion of closed complex to open complex
Repressor binds nonspecifically; slides until it finds the operators
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73. lactose
Inducers
Various β-galactosides can bind repressor and reduce its affinity for the operators 104-fold
Allolactose (produced by action of β-galactosidase on lactose) is a potent inducer
β-galacto- sidase
allolactose
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74. Regulation by cAMP
Cyclic AMP regulatory protein (CRP) has low affinity for DNA in absence of cAMP
With cAMP it becomes an activator that binds just upstream of -35 region and increases transcription
[cAMP] controlled by reductions in extracellular [glucose] via phosphate activation of adenylate cyclase
E.coli CRP mutant 48 kDa homodimer PDB 1HW5, 1.8Å
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75. Post-transcriptional modifications of RNA
Every form of RNA can be modified after transcription
tRNA: initial transcript contains several tRNA precursors
RNAse P breaks that into pieces
RNase D trims 3’ end
tRNA nucleotidyl transferase adds CCA to 3’ end
Covalent modifications of bases
Thermatoga RNase P RNA & protein + tRNA 157kDa total MW PDB 3Q1R, 4.21Å
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76. Modifications of rRNA
Primary transcript in prokaryotes=30S: one copy each of 16S, 23S, 5S rRNAs, plus interspersed tRNAs
5’, 3’ ends of each mature rRNA contain base-paired regions in primary transcript
RNase III binds & cleaves; then further trimming
Eukaryotic processing similar; processed in nucleolus
Aquifex RNase III+dsRNA
66.8 kDa total
PDB 2NUG, 1.7Å
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77. Eukaryotic mRNA processing
Modifications increase stability to cellular exonucleases
5’ end modified during synthesis in nucleus
triphosphate loses one phosphate;
diphosphate reacts with GTP to form a triphosphate + PPi: the “cap”
then the capping guanine gets methylated
CPSF added, polyA polymerase binds, endonuclease acts, polyA tail added
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78. Splicing
Internal sequences (introns) in message removed; remaining pieces are exons
Junctions between those are splice sites
5’ splice site: GURAGU
3’ splice site (branch site): YNYURAY
Probably introns were inserted into genes relatively late in evolution; therefore: little evidence of correlations between structural domains and exons
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79. In the spliceosome
Transesterification: 5’ splice site to branch-site adenylate: makes lariat-shaped intron
Transesterification between 5’ exon and 3’ splice site (becomes joined exon)
Spliceosome keeps products positioned:
> 100 proteins
5 snRNA molecules (~5000 nucleotides)
Each snRNA has proteins associated with it: snRNPs; these are used for other things too
5 kinds of snRNAs: U1, U2, U4-6.
Human Dim2
34kDa dimer PDB 3GIX 1.33Å
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80. Probable sequence of events
Rate-limiting step is intron removal
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81. iClicker question #4
4. Why is repair more important in replication than in transcription?
(a) RNA is less prone to mispairing than DNA
(b) transcription is slow, so it’s less likely to incorporate errors
(c) the errors don’t matter when they happen in the introns
(d) Errors in DNA are persistent; errors in RNA are limited to the immediate transcript.
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