rna metabolism transcription and processing ch353 april 1, 2008
TRANSCRIPT
RNA MetabolismTranscription and Processing
CH353
April 1, 2008
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
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
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
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
Transcription with E. coli RNA Polymerase
Rewinding Unwinding
Template strand
RNA-DNA hybrid ~8 bp
Nontemplate strand
Transcription bubble ~17 bp
Topoisomerase IITopoisomerase I
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
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)
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
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
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
Regulation of Transcription in E. coli
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
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
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)
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
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
Synthesis of Eukaryotic mRNA
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
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
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)
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
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
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
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
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
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
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