1. RNA Metabolism Transcription and Processing
CH353
April 1, 2008

2. 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

3. 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

4. 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

5. 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

6. Transcription with E. coli RNA Polymerase
Topoisomerase I
Topoisomerase II
Transcription bubble ~17 bp
Rewinding
Unwinding
Nontemplate strand
Template strand
RNA-DNA hybrid ~8 bp

7. Properties of E. coli RNA Polymerase
Large complex (Mr 390,000) of 5 core subunits (a2bb’w)
RNA polymerase holoenzyme has additional s subunit required for specific initiation
Most common is s70 (Mr 70,000) for recognition of most promoters; s32 for heat shock promoters; s54 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

8. Promoters for E. coli RNA Polymerase
Recognized by holoenzyme with s70 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)

9. 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: 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); s subunit dissociates after first 8-9 nucleotides are polymerized

10. Transcription with E. coli RNA Polymerase
Termination
Rho (r) 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 (r) dependent
has CA-rich sequences in template and r binding sites on transcript
the r protein has helicase activity; uses ATP for translocation on RNA

11. Regulation of E. coli Transcription
Specific s 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

12. Regulation of Transcription in E. coli

13. 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

14. 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 fractions
Fractions containing TFIIC2 bind labeled VA1 DNA probe, slowing its mobility on non-denaturing PAGE
Yoshinaga et al. (1989) J.Biol.Chem 264, 10726

15. Eukaryotic RNA Polymerases
RNA polymerase I II III
Subunits
unique abb’w-like 5 5* 5
common
unique
Inhibition [a-amanitin] (resistant) low high
Products pre-rRNA mRNAs, tRNAs,
(28S, 5.8S, 18S) snRNAs U6 snRNA,
5S rRNA,
7S RNA
* large subunit has carboxy terminal domain (CTD)

16. Properties of RNA Polymerase II
RBP1, largest subunit (Mr 220,000)
is homologous to E. coli RNA polymerase b’ 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 bp CG-rich region ~100 bp upstream of start
Many have promoter-proximal elements (control regions) within upstream of start

17. 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 nt RNA
Elongation
Elongation factors suppress pausing and coordinate RNA processing;
pTEFb phosphorylates CTD
Termination and Release
Pol II dephosphorylated after release

18. Synthesis of Eukaryotic mRNA

19. 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

20. Splicing of mRNA Transcripts
splice
donor
branch point
splice
acceptor
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

21. 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; ’ OH of intron is leaving group
Products: spliced RNA + intron RNA with 2’,5’ branch (lariat)

22. 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

23. 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

24. 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

25. Splicing of Calcitonin/CGRP mRNA
Precursor mRNA has 2 poly(A) sites:
one recognized in thyroid and other recognized in brain
Shorter thyroid transcript is processed at exons encoding calcitonin – a calcium regulating hormone
Longer brain transcript is processed at exons encoding CGRP (calcitonin gene-related peptide)
– a neurotransmitter
1 gene provides 2 different proteins depending on alternate processing of RNA

26. 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 nucleotides are methylated

27. 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
Site-specific methylation of pre-rRNA using guide RNAs
Nuclease cleavage with:
RNase III
RNase P
RNase E
Processing with various specific nucleases

28. 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