RNA Metabolism

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Chapter 26 RNA Metabolism
Ribosomal RNA, tRNA, mRNA, miRNA.
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BMSE Seminar in Webb 1100 at 3 pm Dr. Frederic
Chedin, UC Davis "De novo DNA methylation in
mammals As easy as A, B, L"
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26.1 DNA-dependent synthesis of RNA Replication
vs transcription RNA polymerases Promoters Tran
scriptional regulation Eukaryotic (nuclear) RNA
polymerases RNA Pol II
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DNA template 4 ribonucleoside 5triphosphates Mg2

5?3 growth No primer needed Transcription
bubble 50-100 nt/s Waves of supercoils
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Adenovirus genome
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Bacterial RNA polymerases (holoenzyme, apoenzyme,
subunits, and transient complexes (s))
No proofreading 1 in 104-105 error rate Why?
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How does RNA polymerase know which genes to
transcribe?
Promoters (-70 to 30) of transcriptional
start Consensus sequences (-10 and -35) s
subunit recognition Evidence (mutational mapping)
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Bacterial transcriptional regulationblend of
negative and positive controls (more negative)
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RNA polymerase core structure
Grey/ß subunits red/a subunits
Remove ß subunit
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Transcription regulation Repressors etc. Vs
eukaryotes
Processivity Sequence-encoded informationterminat
ion ? sites Pausing of RNA Pol RNA/DNA hairpins
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Eukaryotic promoters and polymerases
(I,II,III) Pol I pre-rRNA Pol II mRNA Pol
III tRNAs Distinct promoters
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Transcription at RNA Pol II promoters
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Human TBP.it really bends DNA
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Actinomycin D inhibition of prokaryotic and
eukaryotic RNA Polymerases
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26.2 RNA processing 5 capping of eukaryotic
mRNA Introns and exons RNA catalysis 3 end
structures of eukaryotic mRNAs Differential RNA
processing (splicing0 RNA catalysis revisited
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RNA processing in prokaryotes and eukaryotes is
extensive
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5 Cap of mRNA (eukaryotes)
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The enzyme that does the capping is tethered to
Pol II
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RNA as an enzyme intron splicing and
more Self-splicing Introns predominate in
eukaryotic genome Tetrahymena thermophila (T.
Cech)
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Group I splicing mechanism nucleophile is
guanosine, GMP, GDP, or GTP
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Group II splicing nucleophile is part of intron
and formation of lariat structure
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SpliceosomemRNA primary transcript
synthesis Specialized RNA-protein complexes
(small nuclear ribonucleoproteins (snURPs) GU
and AG at 5 and 3
? pseudouridine
snRNA
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C-terminal domain of RNA Pol II binds components
of splicing complex
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Eukaryotes add a poly (A) tail (80-250 nt) to the
primary RNA transcripts Enzyme complex
includes endonuclease, polyadenylate polymerase,
accessory proteins
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Alternative processing of RNA (splicing
variations) Variations in Cleavage
sites Polyadenylation sites
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Calcitonin-gene-related-peptide
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Bacterial pre-rRNA processing
RNases 1 RNase III 2 RNase P 3 RNase E
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Vertebrate pre-rRNA transcript processing Methylat
ion at 2OH
Cleavage requires snoRNAs
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Processing of tRNAs (bacteria and eukaryotes)
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RNA catalysts Group I introns, RNase P,
Hammerhead ribozyme
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Mg required
Enzyme characteristics Km, kcat/Km
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Size variations of ribozymes. Self-splicing
rRNA intron from Tetrahymena 400 nt
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26.3 RNA-dependent RNA and DNA synthesis Reverse
transcriptase Retroviruses Telomeres and
telomerase RNA-dependent RNA polymerase Evolutio
n
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Reverse transcriptase
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Long terminal repeats ? needed for
packaging Gag interior viral core
proteins Pol protease, integrase, reverse
transcriptase Env viral envelope proteins
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Cancer causing retroviruses Oncogene (e.g., src,
tyrosine-specific kinasecell division,
intracellular communication) Cellular and viral
versions of the same gene
Rous sarcoma virus genome, Bishop/Varmus
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HIV genome
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Transposons, retrotransposons, DNA methylation,
silencing Lack the env gene
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Telomerase, a specialized reverse
transcriptase Telomeres Finishing ends a
problem Generational shortening Telomerase 150
nt RNA Dimeric telomerase Processivity Cancer
therapy, Geron
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Riboswitches
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