Termination of Transcription

Termination of Transcription

• RNA polymerase transcribes until it meets a terminator, then transcription stops and the RNA product dissociates from the DNA template.
• Many terminators are hairpin forming sequences.
• Termination signals in E. coli:
  • Rho-dependent terminators: require a protein factor (Rho)
  • Rho-independent terminators: do not require Rho

Rho-dependent Termination

• Rho Factor:
  • 46-kD protein, active as a hexamer
  • Binds to RNA
  • Has helicase activity, causing RNA-DNA to separate
• Hot pursuit model: Rho binds to RNA tail and moves along transcript until it catches the polymerase

Rho-independent Termination

• Rho-independent terminator:
  • G-C rich stem
  • U-DNA pairing is very weak, allowing dissociation
  • U-run: 7-9 bases after loop

Central Dogma

• Describes the flow of genetic information from DNA to RNA to protein
• Transcription, translation, and degradation of mRNA molecules often occur simultaneously in prokaryotes

Transcription

• Process by which a DNA sequence is copied into an RNA molecule
• RNA polymerase synthesizes the RNA molecule in the 5' to 3' direction using one of the DNA strands as a template

Stages of Transcription

• Initiation: RNA polymerase binds to the promoter region and begins to unzip the DNA strands
• Elongation: RNA polymerase synthesizes the RNA molecule in the 5' to 3' direction using one of the DNA strands as a template
• Termination: RNA polymerase reaches the terminator sequence and stops transcribing the DNA

Differences Between Transcription and DNA Replication

• Transcription produces an RNA molecule, while DNA replication produces a DNA molecule
• Transcription is carried out by RNA polymerase, while DNA replication is carried out by DNA polymerase
• Transcription only requires one strand of DNA as a template, while DNA replication requires both strands of DNA as a template

E.Coli RNA Polymerase

• Core enzyme: α₂ β β' ω
• Holoenzyme: α₂ β β' ωσ
• Functions of the subunits:
  • α: assembly of the tetrameric core
  • β: ribonucleoside triphosphate binding site
  • β': DNA template binding region
  • σ: initiation of transcription

Numbering of a Transcription Unit

• Transcript initiation site is +1
• Bases preceding the initiation site are given minus (-) prefixes and are referred to as upstream sequences
• Bases following the initiation site are given plus (+) prefixes and are referred to as downstream sequences

Promoters

• In E. coli, must be >12 bp to avoid occurrence by chance
• Promoters have only small conservation in sequence
• Four main regions:
  • Startpoint
  • -10 sequence (Pribnow box)
  • -35 sequence
  • 17 nucleotide spacer sequence between -10 and -35 sequences

Starting Transcription

• Binding of RNA polymerase holoenzyme to a promoter region in DNA
• Localized unwinding of both DNA strands by RNA polymerase to provide a single-stranded template
• Formation of phosphodiester bonds between the first few ribonucleotides in the nascent RNA chain
• Conformational change in enzyme, promoter is cleared
• Sigma Factor is released
• Nus A protein binds instead, ready for elongation "antitermination complex"

Elongation

• RNA polymerase is bound to DNA and is covalently extending the RNA chain
• The polymerase moves downstream from its position, processively extending the nascent RNA chain