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RNA Metabolism Transcription and Processing CH353 April 1, 2008.
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Transcript of RNA Metabolism Transcription and Processing CH353 April 1, 2008.
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RNA MetabolismTranscription and Processing
CH353
April 1, 2008
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Types of RNA
Present in all cells, mitochondria and chloroplasts• Messenger RNA (mRNA) – encode protein sequences from DNA• Transfer RNA (tRNA) – decode mRNA sequences; activate specific
amino acids for protein synthesis• Ribosomal RNA (rRNA) – catalytic components of ribosomes; for
tRNA binding, codon-anticodon recognition, peptidyl transfer
Present predominantly in eukaryotic cells • Catalytic RNAs – components of ribonucleoprotein enzymes• 7S RNA – component of signal recognition particle for secretion• Small nuclear RNAs (snRNAs) – spliceosome subunit components• Small nucleolar RNAs (snoRNAs) – guides for rRNA modification• MicroRNAs (miRNAs) – silencing gene expression
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RNA Polymerase Reaction
General reaction: (NMP)n + NTP → (NMP)n+1 + PPi
Template + n NTP → Template + ppp(NMP)n + n PPi
• Requires DNA template, NTP’s and Mg2+ (Initiator NTP is primer)
• Reaction driven by hydrolysis of PPi; ∆G’º = -19 kJ/mol
Mechanism: nucleophilic attack by 3’-OH on α-phosphate of NTP with PPi as leaving group
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Basic Properties of Transcription
• Transcription from DNA without strand separation– Transient transcription bubble of single stranded DNA
• Transcription synthesizes single stranded RNA– Transient RNA-DNA hybrid intermediate
• Transcription is specific to DNA strand– Template strand DNA transcribed; reverse complement of RNA– Nontemplate or coding strand of DNA; same sense as RNA
• Transcription has an initiation site on DNA– RNA synthesis begins at promoters– no primer required: GTP + NTP → (5’) pppGpN-OH (3’) + PPi
• Transcription has a termination site– RNA synthesis ends at terminators; defines DNA template
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Template and Nontemplate Strands
Transcription Units of Adenovirus Genome• Nontemplate sequences same sense as RNA transcripts• Templates for RNA synthesis
– do not include entire DNA strand– could be on either or both DNA strands
• A sequence and its complement do not both encode proteins
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Transcription with E. coli RNA Polymerase
Rewinding Unwinding
Template strand
RNA-DNA hybrid ~8 bp
Nontemplate strand
Transcription bubble ~17 bp
Topoisomerase IITopoisomerase I
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Properties of E. coli RNA Polymerase
• Large complex (Mr 390,000) of 5 core subunits (2’)
• RNA polymerase holoenzyme has additional subunit required for specific initiation
• Most common is 70 (Mr 70,000) for recognition of most promoters; 32 for heat shock promoters; 54 for regulation by enhancers (NtrC)
• RNA polymerases lack 3’ → 5’ exonuclease proofreading activity; can remove incorrect nucleotide by reversal of polymerase reaction
• Error rate: 10-4 to 10-5
• RNA polymerases are highly processive; dissociation of polymerase from DNA terminates transcription
• RNA polymerization rate: 50 – 90 nucleotides / second (comparable to DNA polymerase II); transcription of most genes in < 1 minute
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Promoters for E. coli RNA Polymerase
• Recognized by holoenzyme with 70 subunit (most common)• Alignment of RNA start sites reveals upstream consensus sequences• TTGACA at -35 and TATAAT at -10 relative to start (+1)• Strong promoters have consensus sequences and additional A-T rich
UP element (between -40 and -60)
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Transcription with E. coli RNA Polymerase
Initiation and Elongation• E. coli RNA polymerase holoenzyme binds
to promoter sequence• Closed complex: RNA polymerase bound
but DNA still double stranded• Open complex: 12-15 bp region of DNA
from within -10 to +2 or +3 is unwound• Transcription initiation: formation of full
transcription bubble; conformational change to elongation form
• Elongation form: complex moves away from promoter (promoter clearance); subunit dissociates after first 8-9 nucleotides are polymerized
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Transcription with E. coli RNA Polymerase
Termination• Rho () independent (shown)
– transcription of sequence that can form hairpin loop;
– followed by AAA in template for UUU in transcript
– pausing of RNA polymerase allows hairpin loop formation and disruption of RNA-DNA
• Rho () dependent– has CA-rich sequences in template
and binding sites on transcript– the protein has helicase activity;
uses ATP for translocation on RNA
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Regulation of E. coli Transcription
• Specific subunits determine promoter selection– for generalized changes, e.g. development stage (sporulation) or
stress response (heat shock)
• Activation of transcription (Positive Regulation)– cAMP receptor protein (CRP) involved in catabolite activation;
increases transcription of gene involved in utilization of carbon sources other than glucose
– CRP bound to cAMP binds to DNA upstream of weak promoters
• Repression of transcription (Negative Regulation)– lac and trp repressors block transcription by binding to operator
sequences within or downstream of promoters– lac repressor bound to inducer doesn’t bind to operator
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Regulation of Transcription in E. coli
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DNA Footprinting Analysis
• Used for qualitative analysis of DNA-protein binding and localizing binding sequences
• Uses end-labeled DNA and partial DNase digestion + bound protein
• Partially digested DNA is analyzed by denaturing PAGE
• Sequences binding to protein are protected from DNase digestion – indicated by missing bands on gel
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Electrophoretic Mobility Shift Assay (EMSA)
• Used for quantitative analysis of DNA binding proteins
• DNA fragment containing a promoter region is labeled
• DNA probe incubated with protein fractions suspected of having binding activity
• Assayed fractions are analyzed by non-denaturing PAGE
• Electrophoretic mobility of DNA is slower if bound to a protein
Arrow indicates migration of bound DNA probe
EMSA of protein purification fractionsFractions containing TFIIC2 bind labeled VA1 DNA probe, slowing its mobility on non-denaturing PAGE
Yoshinaga et al. (1989) J.Biol.Chem 264, 10726
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Eukaryotic RNA Polymerases
RNA polymerase I II III
Subunits 14 12 16
unique ’-like 5 5* 5
common 4 4 4
unique 5 3 7
Inhibition [-amanitin] (resistant) low high
Products pre-rRNA mRNAs, tRNAs, (28S, 5.8S, 18S) 5 snRNAs U6 snRNA,
5S rRNA, 7S RNA
* large subunit has carboxy terminal domain (CTD)
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Properties of RNA Polymerase II
• RBP1, largest subunit (Mr 220,000) – is homologous to E. coli RNA polymerase ’ subunit
– has unusual carboxyl-terminal domain (CTD) with heptad (-YSPTSPS-) repeats of 27x (yeast) or 52x (human) plus unstructured linker; CTD extends from main polymerase structure ~ 90 to 160 nm
• RNA polymerase II promoters– Many have TATA box -35 to -26 bp from start (T-A-T-A-A/T-A-A/T-A/G)
– Some have initiator element instead of TATA box (Y-Y-A+1-N-T/A-Y-Y-Y); these also have a 20-50 bp CG-rich region ~100 bp upstream of start
– Many have promoter-proximal elements (control regions) within 100-200 upstream of start
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Transcription with RNA Polymerase II
• Assembly of RNA polymerase and transcription factors at promoter
– Formation of closed complex– TFIIH (12 subunits) has helicase,
kinase and DNA repair activities
• Initiation and promoter clearance– TFIIH phosphorylates CTD of Pol II
causing conformation change initiating transcription; TFIIE, TFIIH released after 1st 60-70 nt RNA
• Elongation– Elongation factors suppress pausing
and coordinate RNA processing; – pTEFb phosphorylates CTD
• Termination and Release– Pol II dephosphorylated after release
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Synthesis of Eukaryotic mRNA
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Structure and Synthesis of mRNA Caps
• Most eukaryotic mRNAs have 5’ cap structure
• 7-methylguanosine with (+) charge linked to 5’-terminal nucleotide of mRNA by 5’,5’ triphosphate
• Unique structure important for translation initiation
• Cap formed by transfer of guanylate to 5’ diphosphate and methylation using S-adenosylmethionine
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Splicing of mRNA Transcripts
Formation of Spliceosome: (5 snRNAs + 50 proteins)
• U1 snRNP binds at splice donor site• U2 snRNP binds at branch point site (+ ATP)• U4:U6 snRNP and U5 snRNP bind forming
inactive splicesome (+ ATP)• U4 snRNP and U1 snRNP released, U6 snRNP
binds splice donor activing spliceosome (+ ATP)• Splicing occurs by 2 step mechanism with lariat
intron and spliced exons as products
splicedonor
spliceacceptor
branch point
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Splicing Reaction Mechanism
Mechanism for spliceosomal introns and self-splicing group II introns
2 step splicing mechanism:• 2’ OH of adenosine at branch point
is nucleophile for attack on splice donor phosphodiester bond; 3’ OH of 5’ exon is leaving group
• 3’ OH of 5’ exon (splice donor) is nucleophile for attack on splice acceptor phosphodiester bond; 3’ OH of intron is leaving group
Products: spliced RNA + intron RNA with 2’,5’ branch (lariat)
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Coupling Transcription and RNA Processing
• C-terminal domain (CTD) provides attachment sites for complexes involved with RNA processing
• Cap-synthesizing complex binds to CTD and 5’ end of mRNA precursor
• 5’ end of RNA is capped• Cap-binding complex binds
CTD and 5’ cap of mRNA• Spliceosome components
bind CTD, capturing splice donors and branch points of nascent mRNA
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Termination and Polyadenylation
• Eukaryotic mRNA have a 3’ terminal poly(A) tail (80 – 250 nucleotides)
• Polyadenylation linked to transcription termination
• Enzyme complex binds CTD cleaving RNA at poly(A) site; between AAUAAA and GU-rich sequences
• Polyadenylate polymerase of enzyme complex adds poly(A) to 3’ OH:
RNA + nATP → RNA-(pA)n + nPPi
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Alternative Splicing of mRNA Transcripts
Alternative Polyadenylation Sites• Poly(A) site choice forms diversity
of 3’ ends for mRNA; C-terminal ends for proteins
Alternative Splicing Patterns• A splice donor may have multiple
splice acceptors, forming diverse mRNAs and encoded proteins
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Splicing of Calcitonin/CGRP mRNA
• Longer brain transcript is processed at exons 1-2-3-5-6 encoding CGRP (calcitonin gene-related peptide) – a neurotransmitter
• 1 gene provides 2 different proteins depending on alternate processing of RNA
• Precursor mRNA has 2 poly(A) sites:– one recognized in thyroid and other recognized in brain
• Shorter thyroid transcript is processed at exons 1-2-3-4 encoding calcitonin – a calcium regulating hormone
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Processing of Human rRNA Precursors
In the nucleolus: • RNA pol I transcribes 45S pre-rRNA from multiple rRNA genes• Small nucleolar RNAs (snoRNA) guide methylation and cleavage of
the precursor into mature 18S, 5.8S and 28S rRNAs• >100 of 14000 nucleotides are methylated
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Processing of Bacterial rRNA Precursors
• E. coli RNA polymerase transcribes RNA from one of 7 rRNA genes• Each precursor contains 16S, 23S and 5S rRNAs plus 1 – 2 tRNAs
1. Site-specific methylation of pre-rRNA using guide RNAs
2. Nuclease cleavage with:1. RNase III
2. RNase P
3. RNase E
3. Processing with various specific nucleases
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Processing tRNA Precursors
• Yeast tRNA is processed from precursor by removing the 5’ end with RNase P (a ribozyme), then the 3’ end with RNase D
• The terminal CCA(3’) is added 1 nucleotide at a time by the enzyme tRNA nucleotidyl transferase – template independent RNA synthesis
• Bases are modified during the processing of tRNA• Some tRNAs are spliced, removing the intron in the last step