Eukaryotic transcription is a complex process that turns DNA into RNA. It involves three main steps: initiation, elongation, and termination. Each step requires specific proteins and enzymes to work together, ensuring accurate gene expression.

RNA polymerase II plays a crucial role in transcribing protein-coding genes. It works with general transcription factors to start transcription, then continues adding nucleotides to form pre-mRNA. The process ends with the addition of a poly(A) tail, preparing the RNA for further processing.

Eukaryotic Transcription

Steps of eukaryotic transcription

1. Initiation

   • Pre-initiation complex (PIC) assembles at the promoter region
     • General transcription factors (GTFs) such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH bind to the promoter along with RNA polymerase II
   • TATA-binding protein (TBP), a subunit of TFIID, recognizes and binds to the TATA box sequence (typically TATAAA) located 25-30 base pairs upstream of the transcription start site
   • Transcription factor IIB (TFIIB) binds to TBP and recruits RNA polymerase II to the promoter
   • Transcription factors IIE (TFIIE) and IIH (TFIIH) join the complex to complete PIC formation
   • TFIIH, which possesses helicase activity, unwinds the DNA template strand to expose the transcription start site and enable RNA polymerase II to initiate transcription
   • The unwound region forms a transcription bubble, allowing RNA polymerase II to access the template strand

2. Elongation

   • RNA polymerase II synthesizes the pre-mRNA molecule in the 5' to 3' direction by adding nucleotides complementary to the DNA template strand
   • Elongation factors such as ELL and Elongin help maintain the stability and processivity of RNA polymerase II during transcription elongation
   • RNA polymerase II continues to synthesize the pre-mRNA until it reaches a termination signal

3. Termination

   • Cleavage and polyadenylation specificity factor (CPSF) recognizes the polyadenylation signal sequence (typically AAUAAA) near the 3' end of the pre-mRNA
   • Cleavage stimulatory factor (CstF) binds to the GU-rich sequence located 20-40 nucleotides downstream of the polyadenylation signal
   • The pre-mRNA is cleaved by the cleavage complex at a site 10-35 nucleotides downstream of the polyadenylation signal
   • Poly(A) polymerase adds a poly(A) tail consisting of 200-250 adenine residues to the 3' end of the cleaved pre-mRNA
   • RNA polymerase II and the associated transcription factors dissociate from the DNA template, marking the end of transcription

Function of RNA polymerase II

• Catalyzes the synthesis of pre-mRNA molecules from protein-coding genes by forming phosphodiester bonds between ribonucleotides
• Maintains the correct reading frame during transcription elongation to ensure accurate base pairing with the DNA template strand
• Interacts with various GTFs, activators, repressors, and elongation factors to regulate the efficiency and fidelity of transcription
• Plays a crucial role in gene expression by producing pre-mRNA molecules that undergo further processing to yield mature mRNA for translation

Roles of RNA polymerases I, II, III

• RNA polymerase I transcribes ribosomal RNA (rRNA) genes in the nucleolus
  • Synthesizes the 28S, 18S, and 5.8S rRNA components of the large and small ribosomal subunits
  • rRNAs are essential for ribosome assembly and function in protein synthesis
• RNA polymerase II transcribes protein-coding genes in the nucleoplasm
  • Produces pre-mRNA molecules that are processed into mature mRNA for translation
  • Also transcribes some small nuclear RNAs (snRNAs) involved in splicing and microRNAs (miRNAs) that regulate gene expression post-transcriptionally
• RNA polymerase III transcribes various small non-coding RNA genes in the nucleoplasm
  • Synthesizes transfer RNAs (tRNAs) that deliver amino acids to ribosomes during protein synthesis
  • Transcribes the 5S rRNA component of the large ribosomal subunit
  • Produces other small RNAs such as U6 snRNA (involved in splicing) and 7SL RNA (a component of the signal recognition particle)

Transcription factors in gene regulation

• General transcription factors (GTFs) are required for the assembly of the pre-initiation complex (PIC) at the promoter region
  • GTFs such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH ensure accurate positioning of RNA polymerase II and facilitate transcription initiation
  • GTFs interact with the core promoter elements (TATA box, initiator, and downstream promoter element) to recruit RNA polymerase II
• Activators enhance the rate of transcription initiation
  • Bind to specific DNA sequences called enhancers located upstream, downstream, or within introns of the regulated gene
  • Interact with co-activators (CBP/p300) and the mediator complex to recruit and stabilize the PIC at the promoter
  • Examples of activators include Sp1, CREB, and NF-κB
• Repressors reduce the rate of transcription initiation
  • Bind to specific DNA sequences called silencers located upstream, downstream, or within introns of the regulated gene
  • Interact with co-repressors (Sin3, NCoR) to inhibit the assembly or stability of the PIC
  • Examples of repressors include REST, YY1, and MeCP2
• Chromatin remodeling factors alter the accessibility of DNA to transcription factors and RNA polymerase II
  • Histone acetyltransferases (HATs) such as Gcn5 and p300/CBP add acetyl groups to lysine residues on histone tails, reducing chromatin compaction and increasing DNA accessibility
  • Histone deacetylases (HDACs) remove acetyl groups from histone tails, promoting chromatin condensation and reducing DNA accessibility
  • ATP-dependent chromatin remodeling complexes (SWI/SNF, ISWI) use energy from ATP hydrolysis to slide or evict nucleosomes, exposing DNA regions for transcription factor binding

Transcription and RNA processing

• The transcription unit encompasses the region of DNA that is transcribed into a single RNA molecule, including the coding sequence and regulatory regions
• Co-transcriptional RNA processing occurs as the pre-mRNA is being synthesized:
  • Capping: Addition of a 7-methylguanosine cap to the 5' end of the pre-mRNA
  • Splicing: Removal of introns and joining of exons to form the mature mRNA
  • Polyadenylation: Addition of a poly(A) tail to the 3' end of the pre-mRNA
• These processes are coordinated with transcription to ensure efficient and accurate production of mature mRNA molecules