Transcription of DNA into RNA, enzymatic reactions, RNA, RNA degradation

  • Transcription
    1. 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.
    2. 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).
    3. 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.
  • 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.

Post-Transcriptional Processing

•Only in the Nucleus for Eukaryotes •Primary transcript processed in 3 ways: –1. ADDition of nucleotides –2. DELETion of nucleotides –3. Modification of Nitrogenous Bases •5’ cap and 3’ Poly A tail •snRNP and the Spliceosome of Doom! –INtrons = IN nucleus –EXtrons = EXit the nucleus


Clone Library = plasmid and replication

Polymerase chain reaction (PCR) : ‘fast’ cloning

•Heat to denature •Mix with primers, let cool = primers hybridize •Add polymerase to amplify complementary strands

Southern Blot

•1. Chop up DNA •2. Efield to spread out pieces by SIZE •3. Blot it! •4. Add Radioactive DNA or RNA Probe •5. Visualize on radiographic film  GENETIC CODE •Degenerative – more than one series of nucleotides may code for ANY A.A. •Unambigous = one series of nucleotides = one A.A. •Universal code! •START! AUG •STOP! UAA, UAG, UGA •4^3 = 64 –Ex. protein of 100 A.A. = there are 20^100 possible amino acids sequences for the protein