Gene Transcription and Expression

Functional organization of a eukaryotic gene

  • Regulation of gene expression
    • Promoter
      • Site where RNA polymerase II and multiple other transcription factors bind to DNA upstream from gene locus (AT-rich upstream sequence with TATA and CAAT boxes).
      • Promoter mutation commonly results in dramatic ↓ in level of gene transcription.
    • Enhancer
      • DNA locus where regulatory proteins (“activators”) bind → increasing expression of a gene on the same chromosome.
      • Enhancers and silencers may be located close to, far from, or even within (in an intron) the gene whose expression it regulates.
    • Silencer
      • DNA locus where regulatory proteins (“repressors”) bind → decreasing expression of a gene on the same chromosome.
  • RNA polymerases
    • Eukaryotes
      • RNA polymerase I makes ribosomal RNA, the most common (rampant) type; transcribe the 45S pre-rRNA gene into a single template that is subsequently processed into mature 18S, 5.8S, and 28S rRNAs. Present only in nucleolus.
      • RNA polymerase II makes:
      • RNA polymerase III makes
        • 5S rRNA (component of 60S ribosomal subunit)
        • tRNA (smallest RNA, tiny).
      • No proofreading function, but can initiate chains. RNA polymerase II opens DNA at promoter site.
      • I, II, and III are numbered in the same order that their products are used in protein synthesis: rRNA, mRNA, then tRNA.
      • α-amanitin, found in Amanita phalloides (death cap mushrooms), inhibits RNA polymerase II. Causes severe hepatotoxicity if ingested.
      • Actinomycin D inhibits RNA polymerase in both prokaryotes and eukaryotes.
    • Prokaryotes
      • 1 RNA polymerase (multisubunit complex) makes all 3 kinds of RNA.
      • Rifampin inhibits DNA-dependent RNA polymerase in prokaryotes.
  • RNA processing (eukaryotes)
    • Initial transcript is called heterogeneous nuclear RNA (hnRNA). hnRNA is then modified and becomes mRNA.
  • The following processes occur in the nucleus:
    • Capping of 5′ end (addition of 7-methylguanosine cap)
    • Polyadenylation of 3′ end (≈ 200 A’s)
      • Poly-A polymerase does not require a template.
      • AAUAAA = polyadenylation signal.
    • Splicing out of introns from pre-mRNA by small nuclear RNA, removes introns containing GU at the 5′ splice site and AG at the 3′ splice site
    • Capped, tailed, and spliced transcript is called mRNA.
    • mRNA is transported out of the nucleus into the cytosol, where it is translated.
    • mRNA quality control occurs at cytoplasmic processing bodies (P-bodies), which contain exonucleases, decapping enzymes, and microRNAs; mRNAs may be degraded or stored in P-bodies for future translation.
  • Introns vs exons
    • Exons contain the actual genetic information coding for protein.
    • Introns are intervening noncoding segments of DNA. Different exons are frequently combined by alternative splicing to produce a larger number of unique proteins.
    • Alternative splicing can produce a variety of protein products from a single hnRNA sequence (eg, transmembrane vs secreted Ig, tropomyosin variants in muscle, dopamine receptors in the brain).
    • Introns are intervening sequences and stay in the nucleus, whereas exons exit and are expressed.
    • Variants in which splicing occurs abnormally are implicated in oncogenesis and many genetic disorders (eg, β-thalassemia, Gaucher disease, Tay-Sachs disease, Marfan syndrome).
  • RNA interference
    • Short (20-30 base pair) non-coding RNA sequence induce post-transcription gene silencing.
    • Silencing RNA: Small interfering (siRNA) and microRNA (miRNA).
    • The human genome encodes >1000 miRNA genes, each one capable of repressing hundreds of target genes. Altered expression of even a few miRNA genes can lead to cellular dysregulation including hematologic and solid malignancies (by silencing an mRNA from a tumor suppressor gene.
    • In addition, synthetic siRNA sequences can be introduced into specific pathogenic genes (eg c-myc oncogene) and are being explored as possible therapeutic agents.
    • After being transcribed, miRNA is processed in the nucleus → forms double-stranded precursor → exported to cytoplasm → cleaved into a short RNA helix by a ribonuclease protein called dicer → strands separated & incorporated into RNA-induced silencing complex (RISC). →  binds complementary sequences to miRNA found on target mRNAs →
      • mRNA degradation (exact match)
      • ribosome and transcription factor do not bind, leading to translation repression (partial match)

 

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