
Transcription of DNA into RNA, enzymatic reactions, RNA, RNA degradation
- Transcription
- Initiation: promoter recognition, closed complex, open complex.
- Promoter:
- Prokaryotic: ←upstream, -35 region, Pribnow box, transcription start site (TSS, +1), downstream→
- Eukaryotic: ←upstream, several upstream elements, TATA box, initiator element containing TSS (+1), downstream→
- The high A-T composition in promoters facilitate unwinding of DNA.
- Template strand = antisense strand = (-) strand = noncoding strand = the DNA strand that serves as the template for transcription.
- Nontemplate strand = sense strand = (+) strand = coding strand = the DNA strand having the same sequence as the transcribed RNA.
- Binding to promoter:
- Prokaryotic:
- holoenzyme = core enzyme (polymerase activity) + σ-subunit (promoter and strand specificity).
- binding first forms the closed complex, and then DNA opens up, forms the open complex.
- Eukaryotic:
- A whole bunch of transcription factors (TFs) involved in promoter recognition, binding, and openning up DNA.
- TBP = Tata binding protein. TAF = TBP associated factor.
- Phosphorylation of Pol II C-terminal domain (CTD) opens DNA up, forms the open complex.
- Polymerase must transcribe using the correct template strand. The σ-factor (prokaryotes) and TFs (eukaryotes) tell the RNA polymerase to bind the coding strand, while using the template strand as the template.
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- Prokaryotic:
- Binding to promoter:
- Promoter:
- Elongation:
- Polymerases:
- Prokaryotes have just one.
- Eukaryotes have three:
- 1. RNA Pol I: makes rRNA (except the small 5S rRNA that resembles a tRNA in size).
- 2. RNA Pol II: makes mRNA.
- 3. RNA Pol III: makes tRNA (and 5S rRNA).
- Incorporation of NTPs.
- Prokaryotes lose σ-subunit. Eukaryotes lose TFs.
- Topoisomerases relaxing supercoils ahead and behind the polymerase.
- Transcription-coupled repair: RNA Pol II encounters DNA damage, backs up, TFIIH comes along, recruits repair enzymes. Defective TFIIH → faulty transcription-coupled repair → Xeroderma pigmentosum and Cockayne syndrome (skin sensitive to sunlight radiation in both diseases).
- Polymerases:
- Termination
- Prokaryotic:
- Intrinsic termination: GC hairpin (stalls polymerase) followed by poly U (slips off).
- Rho-dependent termination: ρ protein catches up to polymerase when it stalls at the hairpin, and bumps it off.
- Eukaryotic:
- Termination consensus sequence reached (AAUAAA).
- Polymerase released somewhere further downstream to the consensus sequence.
- Prokaryotic:
- Initiation: promoter recognition, closed complex, open complex.
- RNA
- 1. RNA = ribonucleic acid, has 2’-OH.
- 2. rRNA = ribosomal RNA
- Most abundant (r for rampant).
- Catalyzes peptide bond formation in the ribosome.
- 3. mRNA = messenger RNA
- Longest (m for massive).
- Contains sequence of codons for translation.
- RNA splicing
- pre-mRNA need to be processed.
- Introns = interfering sequences, cut out.
- Exons = spliced together.
- RNA splicing proceeds via a lariat intermediate, by the action of the spliceosome (snRNPs), introns released in lariat form.
- Some RNA can self splice.
- 4. tRNA = transfer RNA
- Smallest (t for tiny).
- Contains anticodon.
- Shuttles the correct amino acid to the correct codon during translation.
- 5. snRNPs (snurps) = RNA + protein, involved in RNA splicing.
- RNA degradation
- RNases degrade RNA.
- Post-transcriptional modifications protect RNA from degradation (5’ cap and polyA tail)
- 2’-O-methylation prevents that position from attacking the RNA backbone.
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