RNA Metabolism Chap 26, part I

mRNA (selective and regulated)tRNArRNAother (specialized) RNAs (eukaryotes!!!)processingtranscriptome (Surprisingly, much of your genome is transcribed!)

RNA is the only class of biomolecule known to exhibit bothinformation (storage/transmission) and catalytic capabilities. Comment on relationship of this finding to our thoughts aboutearly life forms.

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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) Mg2+ See Fig. 26-1 (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:

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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 thebubble. See Fig. 26-1 (c)

7. RNA polymerase footprint covers (literally), ~35 bp(evidence? see Fig. 2, Box 26-1). Term:footprinting

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RNA pol + DNA W complex

Could you measure a Kd for the interaction with thistype of assay?

From Box 26-1, real footprinting data of the lacpromoter.

The data show that polymerase has 2 binding sites,separated by ~5 bp.

8. Only one (exceptions? Fig. 26-3) of the two strandsis transcribed. Language for strands:

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9. An exception to #8. Fig. 26-3: transcripts producedfrom the adenovirus genome.

Think about what this means for a second. If it doesn’tseem a bit crazy, see the Genetic Code (p. 1107) re.constraints. Comment on political smear: “Keepthrowing at it, and see if anything sticks.”

3-D structure from Thermus aquaticus, Fig. 26-4

Derived from pdb: 1hqm. The file has nice views of theZn2+ and Mg2+ (see next page) binding sites.

What fits in the groove?

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Elongation rate in E. coli . 50 to 90 nucleotides persecond.See: http://www.dnalc.org/resources/3d/13-transcription-advanced.html(Based on some of the description in the video, I think they are modeling eukaryotictranscription. Also, re. globin chain in following video on translation.)

At right is the Mg2+ binding site fromthe Taq RNA polymerase (pdb 1hqm)

In the pdb structures I looked at, Ifound at most one Mg2+ co-crystalizingper RNA polymerase.

Chain designators are a little confusing:The β! subunit is indicated as the Dchain.

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B. RNA synthesis begins at specific sites,promoters.

0. What would be the consequence of random (locationof) intitation of RNA synthesis?

1. Promoters: specific DNA sequences that bind RNApolymerase components with relatively high affinity. If 2 things stick together, something (interactionoptions?) must be holding them near each other.

2. In E. coli RNA polymerase binding (70σ) occurs !70to + 30 bp relative to start site.

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3. Consensus (context dependent?) sequence for E. coli, UP = upstream promoter

Fig. 26-5

a) Should all promoters have the consensus sequence? (Logic?)

b) What experiments could you propose to determine theimportance of the sequences in the !35 and !10 regions?

4. UP (upstream promoter) elements are associated withgenes that are transcribed at very high rates. α-subunit of RNA polymerase binds UP elements.

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5. The overall rate of transcription initiation (initiationefficiency?) appears to be related to the affinity of α-subunit binding to UP regions and σ-subunit bindingto !35 and !10 regions.

Fig. 26-6 summarizes initiation/elongation events:

6. Transcription for specific sets of genes can becontrolled w/ different forms of σ (See Table 26-1).

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C. Transcription is regulated at several levels(more on this in Chap 28)

1. cAMP receptor protein (CRP) (E. coli carbohydratemetabolism) activates transcription (+regulation)

2. Repressors inhibit transcription (!regulation)a) lacb) lambda

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D. Specific sequences signal termination of RNAsynthesis Better understood in prokaryotes.

1. ρ-independent termination. RNAs contain:

a) internally complementary sequenced centered ~ 15-20bases from transcript endHairpin formation. See Fig. 26-7 (a).

b) 3 U residues near the 3'-end of the hairpin.

c) Postulate: RNA polymerase pauses shortly after hairpin iscompleted, and hairpin “helps” RNA dissociate fromDNA.

Fig 26-711

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 terminationsite.

c) Then it facilitates dissociation. (Mechanism? ATPhydrolysis?)

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 ofmRNAs and some other RNAs. Initiationrecognition sites, see Fig. 26-8, TATA box.

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3. RNA polymerase III catalyzes the synthesis of 5SrRNA and some other small RNAs. Some of theDNA sites associated with regulation of RNA pol IIIcatalyzed transcription are located within the gene.

F. RNA polymerase II requires many other proteinfactors for its activity

1. Because of the high level of regulation of mRNAsynthesis, RNA polymerase II must interact withmany regulatory proteins.

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2. RNA polymerase II exhibits strong homology toprokaryotic 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 forinitiation & elongation. Fig. 26-9 & Table 26-2:

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Best way to teach/learn this kind of list? Function vs. name?

5. Steps in eukaryotic RNA synthesisa) RNA polymerase and other factors bind at promoterb) RNA strand synthesis starts & promoter region is clearedc) elongationd) termination and release

Fig. 26-96. Regulation: see Chapt 28

7. TFIIH has some unusual and striking functions(strand asymmetric DNA repair!)(Note: TF = transcription factor)

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G. DNA-dependent RNA polymerase undergoesselective inhibition

1. Actinomycin D & acridine intercalate into DNA. The intercalated DNA complex limits movement ofthe transcription bubble. See Fig. 26-10.

2. Rifampacin inhibits bacterial RNA polymerase (β-subunit interactions) Antibiotic

3. α-Amanitin. Mushroom pickers beware. Inhibitspolymerase II at low concentrations and polymeraseIII at higher concentrations. Not a quick way to die.

Fig. 26-10 pdb: 1dsc

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RNA Metabolism (Chap 26) part II

II. RNA ProcessingSome prokaryotic and most eukaryotic RNAs are post-

transcriptionally modified. Some of these modificationrxns. are catalyzed by RNA enzymes (ribozymes).

Intro: RNA processing1. primary transcript ÷ ÷ ÷ ÷ ÷ ÷ ??? mature

transcript2. mRNA: introns, cap, polyA tail (See Fig. 26-11)3. rRNAs: cleavage4. tRNAs: lots of different covalent modifications

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Fig 26-11 eukaryotic mRNA processing

A. Eukaryotic mRNAs are capped at the 5' end (Fig. 26-13)

1. Protects RNA from ribonucleases (significance?)2. Aids in mRNA binding to ribosome

Fig. 26-12 a) 5' cap

Can you see how this structure might confound a 5' (or3') exonuclease?

Fig. 26-12 b) & c)

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B. Both introns & exons are transcribed into RNA Look back at Fig. 26-11?

1. Bacterial genes: DNA sequence converted linearlyw/out breaks into mRNA.

2. 1977: Eukaryotic genes almost always (not histonesand a few other genes) contain intervening sequences(introns) that must be spliced out to produce maturemRNA.

C. RNA catalyzes the splicing of (?) introns

There are 4 classes of introns. The 1st two are:19

1. Group I: self-splicing, found in nuclear,mitochondrial, and chloroplast genes coding formRNAs, rRNAs, & tRNAs.

2. Group II: self-splicing, found in mitochondrial andchloroplast 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 1st one: Fig. 26-13a) Fig. 26-14: Both steps, Group I intron (from distance): b) Fig. 26-14: Both steps, Group II intron (lariat mechanism)

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4. Most introns are not self-splicing (& thereforenot Group I or II introns). These rxns. are catalyzed by the spliceosome,

composed of RNA-protein complexes: smallnuclear ribonucleoproteins (snRNPs)RNA part is called snRNA. (U1-2 & U4-6).

Spliceosomal introns often have GU at 3' ends. Fig 26-16a

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D. Eukaryotic mRNAs have a distinctive 3' endstructure0. Rxn catalyzed by polyadenylate polymerase

a) Primer: yesb) 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. 26-17

3'-polyA tail addition

3. Eukaryotic mRNA processing summary (ovalbumin) 22

E. A Gene Can Give Rise to Multiple Products byDifferential RNA Processing1. Some eukaryotic transcripts yield 1 mature mRNA2. 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. 26-20)

Summary of splicing patterns (Fig 26-21)

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F. rRNAs and tRNAs Also Undergo Processing(eukaryotes, prokaryotes, and archae)

0. Some examples of covalent alterations (Fig. 26-22):1. rRNAs (see Fig. 26-24 re. bacteria, Fig. 26-25 re. us,

vertebrates)a) Bacterial 16, 23, & 5S rRNA’s from 1 transcript Fig 26-23

methylation uridine º pseudouridine uridine º dihydrouridine

b) Vertebrates small/large subunit bundles from 1 transcript

Fig. 26-24 uridine º dihydrouridine!

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Nucleolus: rRNA transcription, maturation, and ribosomeassembly are coupled.

snoRNAs (small nucleolar RNAs) help with modification andcleavage rxns. There are many!!! ~70 in yeast, probably more inhumans.

snoRNPs (snoRNA-protein complexes) participate inmodification of eukaryotic rRNAs.

snoRNAs guide rRNA modification (site alignment),Fig. 26-25

Because of snoRNA guidance 1 enzyme can modifymany different rRNA sites accurately.

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2. tRNAs: cells usually have 40-50 different tRNAs(proaryotes). For yeast: see Fig. 26-26

Unusual enzyme for CCA-3' modification. (3E struct)

G. Special-Function RNAs Undergo SeveralTypes of ProcessingMicro RNAs (miRNAs) contribute to regulation of

gene expression. Mechanisms:1. mRNA cleavage2. Suppression of transcription (like silencing?)

See Fig. 26-27.

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H. RNA Enzymes are the Catalysts in SomeAspects of RNA Processing

1) Group I introns (can function as true catalysts, butare degraded quickly in vivo)

2) RNase P (RNA-protein complex, but the RNA cando the catalysis alone)a) M1 RNA 377 nucleotides longb) protein 17,500 MW (? aa’s long?)

3) Hammerhead ribozymes (self-cleavage)

4) L-19 IVS: intron derived RNA polymerase (sort of)

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I. Cellular mRNAs: Degraded at Different Rates1. Average half-life for vertebrate mRNA ~3 hrs.2. Range: Seconds to many hours3. 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 RandomSequence RNA-like Polymers (no template).

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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 cDNAlibraries are an important facet of their current use.

Comment: applications of what was once basic science!!! http://books.google.com/books?hl=en&lr=&id=ZzDgiqZDX28C&oi=fnd&pg=PA129&dq=history+of+retroviral+research+Temin+Baltimore&ots=uf_svXe1OG&sig=wkX9T8q4CRMTMRhHhgqHR3griUs#v=onepage&q&f=false

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Revised Central Dogma Fig. 26-31

Reverse transcriptases catalyze 3 distinct rxns.:1. RNA-dependent DNA synthesis2. RNA degradation (RNAses)3. DNA-dependent DNA synthesis

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B. Some retroviruses cause cancer and AIDS1. Rous sarcoma virus (see Fig. 26-34)2. HIV and AIDS (see Fig. 26-35)

Comments on logical approaches to combating disease.

See also Box 26-2.

Back to the enzymes chapter: What does KM tell you?

AZT KM for HIV reverse transcriptase is much lowerthan that for human DNA polymerase.

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C. Many Retroviruses, Transposons & IntronsMay Have a Common Evolutionary Origin1. DNA transposons share some structural features

with retroviruses. Relates to use of the term“retrotransposons.” Code for an enzymehomologous to reverse transcriptase. Also LTR sequences. Two eukaryotic examples:

2. Some group I & II introns are mobile geneticelements. 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 externalsource.

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d) Group I intron process occurs at the DNA level (homing),whereas the group II process occurs at the RNA level(retro-homing).

Fig. 26-37 c)

D. Telomerase: Specialized ReverseTranscriptase

Fig. 26-38

E. Some Viral RNAs Are Replicated by RNA-Dependent RNA Polymerase (pro- & eukaryotic)

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F. RNA Synthesis Offers Important Clues toBiochemical Evolution

Fig. 26-40 a)

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