ch 13

• RNA serves as the intermediate molecule between DNA and proteins
• RNA is synthesized on a DNA template during transcription
• RNA polymerase


  Directs the synthesis of RNA using a DNA template
• No primer is required for initiation
• RNA polymerase uses ribonucleotides instead of deoxyribonucleotides
• E. Coli RNA Polymerase

  • Holoenzyme with multiple subunits: a (2 copies), b, b', w (omega) and s (sigma)
  • Core enzyme is a2bb' w
  • Holoenzyme is Core + s factor
  • Sigma plays a regulatory function in the initiation of RNA transcription
• Promoters


  Specific DNA sequences in 5′ region upstream of initial transcription point
• Transcription begins with template binding by RNA polymerase at promoter
• Transcription results in ssRNA
• Template strand is transcribed
• Nontemplate strand is similar to transcribed RNA. AKA coding strand
• Consensus sequence


  Most frequent sequence at that site in different genes
• E. coli promoters


  • Have two consensus sequences: TATAAT (Pribnow box) positioned at -10 from the site of transcription initiation, and TTGACA positioned at –35
  • Degree of binding depends on the strength of the promoter
  • Strength of the promoter = how close the sequence resembles the consensus sequence
• Sigma subunit


  Responsible for promoter recognition
• After ~8 nucleotides, sigma factor dissociates and chain elongation proceeds under the core enzyme
• Bacterial Transcription Termination


  • Intrinsic (Rho-independent) and Rho-dependent
  • Intrinsic termination: Transcribed RNA folds back on itself (hairpin)
  • Rho-dependent termination: Requires the termination factor, rho (r)
• Differences in transcription between prokaryotes and eukaryotes

  • Location in the cell: cytoplasm (prokaryotes) vs nucleus (eukaryotes)
  • Co-Translation: yes (prokaryotes) vs no (eukaryotes)
  • Processing: limited (prokaryotes) vs extensive (eukaryotes)
  • Major RNA polymerases: 1 (prokaryotes) vs 3 (eukaryotes)
  • Holoenzyme RNAP sufficient?: yes (prokaryotes) vs no (eukaryotes)
• Transcription in eukaryotes occurs in the nucleus
• Eukaryotic transcription is not coupled to translation (exception: mitochondria)
• Eukaryotic transcription requires chromatin remodeling
• Eukaryotic RNA polymerases rely on transcription factors (TFs) to scan/bind DNA
• In addition to promoters, enhancers and silencers also influence transcription regulation in eukaryotes
• Eukaryotic RNA transcripts are extensively processed
• Eukaryotic RNA Polymerases


  • RNA Pol II is responsible for transcription of wide range of genes in eukaryotes
  • Activity of RNAP II is dependent on cis-acting elements and trans-acting transcription factors
  • RNAP II core-promoter determines where RNAP II binds to DNA
• Eukaryotic Promoter


  • Regulatory sequences influence efficiency of transcription initiation by RNAP II
  • Four cis elements: Core promoter, Proximal-promoter elements, Enhancers, Silencers
  • TATA box is part of the core promoter, positioned -30 from the transcription start site, consensus: TATA A/T AAR, bound by TATA-binding protein (TBP) of transcription factor TFIID
• Eukaryotic Transcription Factors


  • GENERAL - absolutely required for RNAP II to bind

  SPECIFIC - influence the efficiency or rate of RNAP II binding
• Eukaryotic mRNAs require processing to produce mature mRNAs
• Eukaryotic mRNA Processing


  • Addition of 5' cap

  3' polyadenylation

  RNA splicing

  RNA Editing
• 5' Cap


  1. methylguanosine added shortly after RNA synthesis has begun, protects mRNA from nucleases, facilitates transport and translation initiation
• 3' Polyadenylation


  Stretch of up to ~250 adenosine residues added at the 3'-end, protects mRNA from degradation, important for export and translation
• Introns and Exons


  Introns are regions of initial RNA transcript not expressed in amino acid sequence, exons are sequences retained and expressed

  Prokaryotes do not have introns
• Splicing


  • Two general mechanisms: Self-splicing and Spliceosome-mediated

  Self-splicing: Group I and Group II self-splicing introns, RNA-enzyme (ribozyme)

  Spliceosome: More common, large ribonucleoprotein complex, two transesterification reactions
• RNA Editing


  Substitution editing: Identities of individual nucleotide bases are altered

  Insertion/deletion editing: Nucleotides are added/deleted from total number of bases
• Electron microscopy and interpretive drawing of transcription in E. coli shows RNA strands emanating from different points along the template, with multiple ribosomes bound to an mRNA (polysomes)
• Octopus and squid neurons have insertion/deletion editing
• Insertion/deletion editing


  Nucleotides are added/deleted from total number of bases
• Insertion/deletion editing is prevalent in mitochondrial and chloroplast RNAs
• Substitution Editing: APOBEC and ADAR

  I (inosine) behaves like a G
• Electron microscopy and interpretive drawing of transcription in E. coli shows RNA strands emanating from different points along template—numerous transcription events are occurring simultaneously
• In prokaryotes and eukaryotes, multiple ribosomes bound to an mRNA are called polysomes (or polyribosomes)