RNA Metabolism Chap 26, part I

Transcription

1 RNA Metabolism Chap 26, part I mrna (selective and regulated) trna rrna other (specialized) RNAs (eukaryotes!!!) processing transcriptome (Surprisingly, much of your genome is transcribed!) RNA is the only class of biomolecule known to exhibit both information (storage/transmission) and catalytic capabilities. Comment on relationship of this finding to our thoughts about early life forms. 1

2 I. DNA-Dependent RNA Synthesis Similar to DNA synthesis in some ways. (initiation, elongation, termination) A. RNA is synthesized by RNA polymerases 1. Requirements (substrates?) a) DNA template Fig 26-1(b) b) XTPs (monomers) c) Mg 2+ See Fig (a) 2. A primer is not required for RNA synthesis. 3. Only one strand is transcribed (Exceptions do exist. Fig. 26-3) High selective pressure on genome size?) Adenovirus: 2

3 Fig 26-1(a) Fig. 26-1(b): Template, yes; primer, no. 4. Unwind the double helix 5. RNA polymerase binds to form transcription bubble 6. Movement of transcription bubble generates + supercoiling ahead and!supercoiling after the bubble. See Fig (c) 7. RNA polymerase footprint covers (literally), ~35 bp (evidence? see Fig. 2, Box 26-1). Term: footprinting 3

4 RNA pol + DNA W complex Could you measure a K d for the interaction with this type of assay? From Box 26-1, real footprinting data of the lac promoter. The data show that polymerase has 2 binding sites, separated by ~5 bp. 8. Only one (exceptions? Fig. 26-3) of the two strands is transcribed. Language for strands: 4

5 9. An exception to #8. Fig. 26-3: transcripts produced from the adenovirus genome. Think about what this means for a second. If it doesn t seem a bit crazy, see the Genetic Code (p. 1107) re. constraints. Comment on political smear: Keep throwing at it, and see if anything sticks. 3-D structure from Thermus aquaticus, Fig Derived from pdb: 1hqm. The file has nice views of the Zn 2+ and Mg 2+ (see next page) binding sites. What fits in the groove? 5

6 Elongation rate in E. coli. 50 to 90 nucleotides per second. See: (Based on some of the description in the video, I think they are modeling eukaryotic transcription. Also, re. globin chain in following video on translation.) At right is the Mg 2+ binding site from the Taq RNA polymerase (pdb 1hqm) In the pdb structures I looked at, I found at most one Mg 2+ co-crystalizing per RNA polymerase. Chain designators are a little confusing: The β! subunit is indicated as the D chain. 6

7 B. RNA synthesis begins at specific sites, promoters. 0. What would be the consequence of random (location of) intitation of RNA synthesis? 1. Promoters: specific DNA sequences that bind RNA polymerase components with relatively high affinity. If 2 things stick together, something (interaction options?) must be holding them near each other. 2. In E. coli RNA polymerase binding ( 70 σ) occurs!70 to + 30 bp relative to start site. 7

8 3. Consensus (context dependent?) sequence for E. coli, UP = upstream promoter Fig a) Should all promoters have the consensus sequence? (Logic?) b) What experiments could you propose to determine the importance of the sequences in the!35 and!10 regions? 4. UP (upstream promoter) elements are associated with genes that are transcribed at very high rates. α- subunit of RNA polymerase binds UP elements. 8

9 5. The overall rate of transcription initiation (initiation efficiency?) appears to be related to the affinity of α- subunit binding to UP regions and σ-subunit binding to!35 and!10 regions. Fig summarizes initiation/elongation events: 6. Transcription for specific sets of genes can be controlled w/ different forms of σ (See Table 26-1). 9

10 C. Transcription is regulated at several levels (more on this in Chap 28) 1. camp receptor protein (CRP) (E. coli carbohydrate metabolism) activates transcription (+regulation) 2. Repressors inhibit transcription (!regulation) a) lac b) lambda 10

11 D. Specific sequences signal termination of RNA synthesis Better understood in prokaryotes. 1. ρ-independent termination. RNAs contain: a) internally complementary sequenced centered ~ bases from transcript end Hairpin formation. See Fig (a). b) 3 U residues near the 3'-end of the hairpin. c) Postulate: RNA polymerase pauses shortly after hairpin is completed, and hairpin helps RNA dissociate from DNA. Fig

12 2. ρ-dependent termination. Involves: a) CA rich sequence (rut for rho utilization) b) ρ-protein binds to nascent RNA and migrates along the strand until it reaches a bubble paused at the termination site. c) Then it facilitates dissociation. (Mechanism? ATP hydrolysis?) E. Eukaryotic cells: 3 diff. nuclear RNA polymerases 1. RNA polymerase I: only the synthesis of pre-rrna. 2. RNA polymerase II catalyzes the synthesis of mrnas and some other RNAs. Initiation recognition sites, see Fig. 26-8, TATA box. 12

13 3. RNA polymerase III catalyzes the synthesis of 5S rrna and some other small RNAs. Some of the DNA sites associated with regulation of RNA pol III catalyzed transcription are located within the gene. F. RNA polymerase II requires many other protein factors for its activity 1. Because of the high level of regulation of mrna synthesis, RNA polymerase II must interact with many regulatory proteins. 13

14 2. RNA polymerase II exhibits strong homology to prokaryotic RNA polymerases. a) RBP1 to the β subunit of prokaryotic RNA polymerase. b) RBP2 to the β subunit. c) RBP3 and 11 to show some homology to the prokaryotic α-subunit. 3. Unusual structure at carboxyl terminal tail domain (CTD) region: -YSPTSPS- separated by linker a) 27 repeats in yeast (18 exact matches to consensus seq.) b) 52 repeats in humans & mice (21 exact) 4. Binding of many other protein factors is required for initiation & elongation. Fig & Table 26-2: 14

15 Best way to teach/learn this kind of list? Function vs. name? 5. Steps in eukaryotic RNA synthesis a) RNA polymerase and other factors bind at promoter b) RNA strand synthesis starts & promoter region is cleared c) elongation d) termination and release Fig Regulation: see Chapt TFIIH has some unusual and striking functions (strand asymmetric DNA repair!) (Note: TF = transcription factor) 15

16 G. DNA-dependent RNA polymerase undergoes selective inhibition 1. Actinomycin D & acridine intercalate into DNA. The intercalated DNA complex limits movement of the transcription bubble. See Fig Rifampacin inhibits bacterial RNA polymerase (βsubunit interactions) Antibiotic 3. α-amanitin. Mushroom pickers beware. Inhibits polymerase II at low concentrations and polymerase III at higher concentrations. Not a quick way to die. Fig pdb: 1dsc 16

17 RNA Metabolism (Chap 26) part II II. RNA Processing Some prokaryotic and most eukaryotic RNAs are posttranscriptionally modified. Some of these modification rxns. are catalyzed by RNA enzymes (ribozymes). Intro: RNA processing 1. primary transcript??? mature transcript 2. mrna: introns, cap, polya tail (See Fig ) 3. rrnas: cleavage 4. trnas: lots of different covalent modifications 17

18 Fig eukaryotic mrna processing A. Eukaryotic mrnas are capped at the 5' end (Fig ) 1. Protects RNA from ribonucleases (significance?) 2. Aids in mrna binding to ribosome Fig a) 5' cap Can you see how this structure might confound a 5' (or 3') exonuclease? Fig b) & c) 18

19 B. Both introns & exons are transcribed into RNA Look back at Fig ? 1. Bacterial genes: DNA sequence converted linearly w/out breaks into mrna : Eukaryotic genes almost always (not histones and a few other genes) contain intervening sequences (introns) that must be spliced out to produce mature mrna. C. RNA catalyzes the splicing of (?) introns There are 4 classes of introns. The 1 st two are: 19

20 1. Group I: self-splicing, found in nuclear, mitochondrial, and chloroplast genes coding for mrnas, rrnas, & trnas. 2. Group II: self-splicing, found in mitochondrial and chloroplast genes coding for mrnas. Both of these may also be found (rarely) in bacteria. 3. Group I & II splicing doesn t require ATP, & involves 2 transesterification rxns. in mechanism. Detail on the 1 st one: Fig a) Fig : Both steps, Group I intron (from distance): b) Fig : Both steps, Group II intron (lariat mechanism) 20

21 4. Most introns are not self-splicing (& therefore not Group I or II introns). These rxns. are catalyzed by the spliceosome, composed of RNA-protein complexes: small nuclear ribonucleoproteins (snrnps) RNA part is called snrna. (U1-2 & U4-6). Spliceosomal introns often have GU at 3' ends. Fig 26-16a 21

22 D. Eukaryotic mrnas have a distinctive 3' end structure 0. Rxn catalyzed by polyadenylate polymerase a) Primer: yes b) Template: no 1. Poly A tail decreases rate of RNA degradation. 2. Some prokaryotic mrnas can be modified with a poly A tail. Those show enhanced degradation rates. Fig '-polyA tail addition 3. Eukaryotic mrna processing summary (ovalbumin) 22

23 E. A Gene Can Give Rise to Multiple Products by Differential RNA Processing 1. Some eukaryotic transcripts yield 1 mature mrna 2. Some produce multiple mature transcripts a) Alternative termination points (26-19 a) b) Alternative splicing patterns (26-19 b) Reading frame conserved? Some of these are tissue specific (Fig ) Summary of splicing patterns (Fig 26-21) 23

24 F. rrnas and trnas Also Undergo Processing (eukaryotes, prokaryotes, and archae) 0. Some examples of covalent alterations (Fig ): 1. rrnas (see Fig re. bacteria, Fig re. us, vertebrates) a) Bacterial 16, 23, & 5S rrna s from 1 transcript Fig methylation uridine º pseudouridine uridine º dihydrouridine b) Vertebrates small/large subunit bundles from 1 transcript Fig uridine º dihydrouridine! 24

25 Nucleolus: rrna transcription, maturation, and ribosome assembly are coupled. snornas (small nucleolar RNAs) help with modification and cleavage rxns. There are many!!! ~70 in yeast, probably more in humans. snornps (snorna-protein complexes) participate in modification of eukaryotic rrnas. snornas guide rrna modification (site alignment), Fig Because of snorna guidance 1 enzyme can modify many different rrna sites accurately. 25

26 2. trnas: cells usually have different trnas (proaryotes). For yeast: see Fig Unusual enzyme for CCA-3' modification. (3E struct) G. Special-Function RNAs Undergo Several Types of Processing Micro RNAs (mirnas) contribute to regulation of gene expression. Mechanisms: 1. mrna cleavage 2. Suppression of transcription (like silencing?) See Fig

27 H. RNA Enzymes are the Catalysts in Some Aspects of RNA Processing 1) Group I introns (can function as true catalysts, but are degraded quickly in vivo) 2) RNase P (RNA-protein complex, but the RNA can do the catalysis alone) a) M1 RNA 377 nucleotides long b) protein 17,500 MW (? aa s long?) 3) Hammerhead ribozymes (self-cleavage) 4) L-19 IVS: intron derived RNA polymerase (sort of) 27

28 I. Cellular mrnas: Degraded at Different Rates 1. Average half-life for vertebrate mrna ~3 hrs. 2. Range: Seconds to many hours 3. Average mrna pool turns over ~10 times per cell cycle. (How long is a cell cycle?) 4. Average half-life for bacterial mrna ~very short. J. Polynucleotide Phosphorylase Makes Random Sequence RNA-like Polymers (no template). 28

29 RNA Metabolism (Chap 26) part III III. RNA-Dependent Synthesis of RNA & DNA A. Reverse transcriptase produces DNA from (viral) RNA Much work was initially on retroviruses systems, but cdna libraries are an important facet of their current use. Comment: applications of what was once basic science!!! +retroviral+research+temin+baltimore&ots=uf_svxe1og&sig=wkx9t8q4crmtmrhhhgqhr3gri Us#v=onepage&q&f=false 29

30 Revised Central Dogma Fig Reverse transcriptases catalyze 3 distinct rxns.: 1. RNA-dependent DNA synthesis 2. RNA degradation (RNAses) 3. DNA-dependent DNA synthesis 30

31 B. Some retroviruses cause cancer and AIDS 1. Rous sarcoma virus (see Fig ) 2. HIV and AIDS (see Fig ) Comments on logical approaches to combating disease. See also Box Back to the enzymes chapter: What does K M tell you? AZT K M for HIV reverse transcriptase is much lower than that for human DNA polymerase. 31

32 C. Many Retroviruses, Transposons & Introns May Have a Common Evolutionary Origin 1. DNA transposons share some structural features with retroviruses. Relates to use of the term retrotransposons. Code for an enzyme homologous to reverse transcriptase. Also LTR sequences. Two eukaryotic examples: 2. Some group I & II introns are mobile genetic elements. a) They self-splice. b) They code for an endonuclease that promotes movement. c) One outcome is insertion of intron into homologous DNA (lacking the intron) introduced from some external source. 32

33 d) Group I intron process occurs at the DNA level (homing), whereas the group II process occurs at the RNA level (retro-homing). Fig c) D. Telomerase: Specialized Reverse Transcriptase Fig E. Some Viral RNAs Are Replicated by RNA- Dependent RNA Polymerase (pro- & eukaryotic) 33

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35 F. RNA Synthesis Offers Important Clues to Biochemical Evolution Fig a) 35