Prokaryotic Transcription Overview

Prokaryotic Transcription

• Sigma factor is a subunit of RNA polymerase that binds to the core enzyme, forming the holoenzyme.
• RNA polymerase holoenzyme can bind to DNA, specifically to the promoter sequence.
• Promoter sequences include the -35 and -10 sequences, recognized by the sigma factor.
• -10 sequence is AT-rich, allowing for DNA strand separation to form an open complex.
• Different genes have different -35 and -10 sequences due to the use of different sigma factors.
• Point mutations in the promoter sequence can increase gene expression if they are similar to the consensus sequence.

Initiation of Transcription

• RNA polymerase core is a heteropentamer consisting of 5 subunits.
• Sigma factor binds to the core, forming the RNA polymerase holoenzyme.
• Holoenzyme scans DNA for the -35 and -10 sequences, forming a closed complex.
• DNA strand separation at the -10 sequence creates an open complex.
• Abortive initiation occurs when RNA polymerase binds and unbinds repeatedly due to a lack of primers, leading to unstable binding at the template.

Elongation of Transcription

• Promoter clearance occurs after RNA synthesis begins and RNA polymerase leaves the promoter.
• Sigma factor is released and recycled.
• Transcription termination can occur through two mechanisms:
  • Rho-independent termination: relies on RNA secondary structure and adenine repeats to form a hairpin loop.
  • Rho-dependent termination: relies on the Rho protein binding to a specific site and separating the RNA from the template.

Eukaryotic Transcription

• Eukaryotic transcription is similar to prokaryotic transcription but with several differences:
  • Multiple RNA polymerases: eukaryotes have three RNA polymerases (I, II, and III) compared to one in prokaryotes.
  • More regulatory sequences: eukaryotes have more complex regulatory sequences.
  • Nucleosomes: eukaryotes have nucleosomes that can influence transcription.
  • Transcription factors: eukaryotes require more proteins for initiation.

Transcription Factors

• Transcription factors bind to promoters and affect transcription rates.
• General transcription factors (GTFs): essential for transcription and include TFIID, TFIIB, TFIIF, TFIIE, and TFIIH.
• Regulatory transcription factors: influence transcription rates and include activators and repressors.

Eukaryotic Transcription Initiation Complex

• TFIID: binds to the TATA box via TBP and TAF subunits, bending DNA and recruiting TFIIB.
• TFIIB: helps recruit RNA polymerase II and binds TBP and BRE.
• TFIIF: binds RNA polymerase II and stabilizes TFIIB-pol II interactions.
• TFIIE: recruits TFIIH.
• TFIIH: facilitates initiation to elongation transition through helicase and kinase activity.

Transition from Initiation to Elongation

• Preinitiation complex: formed by five GTFs and the core promoter.
• Initiation complex: formed after TFIIH performs its functions.
• TFIIH triggers the transition by phosphorylating the CTD of RNA Polymerase II, resulting in release of TFIIB, TFIIE, and TFIIH.

Mediator

• Mediator facilitates long-range interactions between regulatory sequences and the promoter region.
• Mediator is located on the tail of RNA polymerase II after phosphorylation by TFIIH.

RNA Processing

• 5' cap: a modified guanine base at the 5' end of RNA that protects the RNA from degradation and facilitates translation, nuclear transport, and splicing.
• 5' cap addition: involves three enzymes:
  • RTPase: removes a phosphate from the 5' end.
  • GTase: adds GMP to the 5' end.
  • Guanylyl-7-methyltransferase: adds a methyl group to create 7-methyl-guanosine.

3' Polyadenylation

• 3' polyadenylation: adds a poly-A tail to the 3' end of RNA that protects the RNA from degradation and serves as a binding site for proteins.
• Polyadenylation process:
  • Polyadenylation signal (AAUAAA): recognized by poly-A binding proteins.
  • Endonuclease: cleaves after the signal sequence.
  • Polyadenylated polymerase (PAP): adds adenine repeats.
  • Poly-A binding proteins: bind to the adenine repeats and protect the 3' end.

RNA Splicing

• RNA splicing: removal of introns and exons from the primary RNA transcript.
• Introns: non-coding sequences within genes.
• Exons: coding sequences within genes.

Spliceosome

• snRNPs (small nuclear ribonucleoproteins): RNA-protein complexes involved in splicing.
• snRNAs: RNA component of snRNPs that help identify splice site sequences.
• Spliceosome: complex formed by snRNPs that catalyzes splicing.
• Splice sites: regions within pre-RNA transcripts where splicing occurs.

Splicing Process

• 5' splice site: GU
• Branch point: an A near the 3' end.
• 3' splice site: AG
• Lariat formation: 2'-OH of the branch point A attacks the phosphate at the 5' splice site, forming a loop.
• Intron removal: the lariat is removed.
• Exon joining: two exons are joined together.