Transcription

• Genes encode proteins and proteins dictate cell function.
• If all cells have the same genetic material; what makes a muscle cell and what makes a red blood cell?

  Differential gene expression regulated by the combination of different transcription factors in that cell, determine cell differentiation and identity/function.
• How is gene expression regulated?

  Primary control point is at the beginning of the protein production process- the initiation of transcription of a gene (transcription makes an efficient control point because many proteins can be made from a single mRNA molecule).
• Cells have distinct sets of transcription regulators; some of these regulators work to increase transcription, whereas others prevent or suppress it, such that only a fraction of the genes in a cell are expressed at any one time.
• Prokaryotes: E.coli core RNA polymerase has 5 polypeptide subunits: B', B, a2 (two copies) and w.

  Holo RNA polymerase: Core + sigma factor (ō).
• Transcription initiation in prokaryotes:

  ~RNA polymerase can bind to DNA non-specifically, but can not recognise the promoter.

  ~Holo enzyme can recognise and bind to the promoter. Sigma factor binds consensus sequence in the core promoter (-35 and -10 sequence elements).
• Transcription initiation in prokaryotes:

  ~Open complex formation: Promoter opening (single stranded DNA region) allows for exposure of the template strand for complementary RNA synthesis.

  
  

  ~Anchored transcription: Only short (abortive) transcripts can be synthesised whilst sigma factor remains bound to the promoter.

  
  

  ~Promoter 'escape' by the RNA polymerase: Sigma factor is released from the promoter. Elongation factor (s) bind and transcription proceeds. The enzyme becomes processive and makes longer transcripts.
• Transcription termination in prokaryotes:

  ~Rho-independent termination: A terminating hairpin forms on the nascent mRNA interacting with the NusA protein to stimulate release of the transcript from the RNA polymerase complex.

  ~The palindromic sequence in the RNA forms a hairpin loop, causing the RNA polymerase to stall.

  ~The U-rich stretch downstream of the termination signal allows the transcript to fall off the template, thereby terminating transcription.
• Transcription termination in prokaryotes:

  ~The Rho protein (RNA helicase) binds at the upstream rut site, translocates down the mRNA, and interacts with the RNA polymerase complex to stimulate release of the transcript.
• In prokaryotes the coding genes are continuous and so transcript (RNA) co-linear with the gene (hybridisation with entire template DNA strand). There is full complementarity.
• In prokaryotes, transcription and translation occur at the same time in the cytoplasm.

  ~The DNA sequence is read in the same order as the amino acid sequence.
• In eukaryotes transcription happens in the nucleus and translation in the cytoplasm.

  ~Noncoding introns within coding sequences.

  ~Introns spliced out; 5' cap and 3' poly A added.
• RNA polymerases in the three kingdoms of life are structurally and functionally conserved.

  ~Bacteria- 5 subunits.

  ~Eukaryotes- 12-17 subunits.
• ~RNA polymerase I transcribes 5.8S, 18S and 28S rRNA genes.

  ~RNA polymerase II transcribes all protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes, IncRNA genes, and most snRNA genes.

  ~RNA polymerase III transcribes tRNA genes, 5S rRNA genes, some snRNA genes, and genes for other small RNAs.
• Core promoter- recognised by general (basal) transcription factors that recruit RNA polymerase.
• Promoter proximal and distal elements: regulatory sequences and binding sites for transcriptional activators and repressors (sequence-specific transcription factors).
• 1. General transcription factors at core promoter (eukaryotes).

  ~RNA polymerase itself cannot find promoters.

  ~TFIID makes multiple specific contacts with core promoter elements, such as TBP (TATA box Binding Protein) interacting with the TATA box.

  ~TFIIB is critical for the recruitment of RNA Polymerase II (with TFIIF).
• 2. General transcription factors at core promoter (eukaryotes).

  
  

  ~This complex can not initiate transcription, even though RNA polymerase II is there.

  ~Once TFIIE and TFIIH have joined a transcription initiation competent pre-initiation complex has formed. They stimulate and stabilise promoter opening, to allow for invitation of transcription by RNA polymerase.