Transcription and RNA Processing Study Notes

Apology for the fast pace of lectures due to the need to finish transcription and RNA processing.
Information on lecture slides on RNA processing published.
Content not covered in class will not appear in the exam.

Definition of Footprint Assay: Technique to determine where transcription factors bind to the DNA, creating a 'footprint.'
Focus on initiation of transcription.

Role of Sigma Factor:
- Binds to RNA polymerase and promoter.
- Forms a closed complex with the DNA outside the polymerase.
- Parts occupy RNA exit channel and DNA entry channel.
- Transcription cannot occur in this state.
Closed Complex to Open Complex:
- Spontaneous isomerization changes shape; this does not require ATP.
- Leads to the formation of an open complex, allowing DNA entry into the polymerase and forming a transcription bubble.
- Applies to σ70 family sigma factors.
- σ54 requires ATPase action, making the isomerization reaction non-reversible but ultimately involves similar steps as σ70 family.

Contrast with DNA Polymerization:
- No primer is required for RNA polymerization.
Abortive Initiation:
- Initial nucleotides form weak bonds, often leading to detachment of the RNA polymerase.
- Requires strong binding between RNA polymerase and sigma factor for stability.
- Successful initiation leads to polymerization of 8-10 nucleotides, allowing elongation to commence.

Sigma Factor and Elongation:
- As RNA strand lengthens, it exits through the RNA exit channel, displacing the sigma factor.
- Once released, the RNA polymerase detaches from sigma factor and can transcribe RNA at a high rate of 50-90 nucleotides per second.
Error Rates in RNA Polymerization:
- Error rate is approximately 1 in 10,000 to 1 in 100,000.
- Lack of exonuclease activity means no proofreading mechanism exists in RNA polymerases.

Two Mechanisms of Termination:
1. Rho-independent Termination:
- Involves a TTT repeat followed by a hairpin structure in the RNA.
- Hairpin formation disrupts the RNA-DNA hybrid, causing detachment.
1. Rho-dependent Termination:
- Relies on a rho utilization site where the rho helicase binds.
- The helicase moves faster than the RNA polymerase, resulting in mechanical disruption and transcription termination.

More complex than prokaryotic transcription with unique regulatory mechanisms.
Utilizes specific transcription factors in addition to general transcription factors.
Enables fine tuning at both gene and individual levels.

Three Different Polymerases:
1. RNA Pol I: Responsible for large ribosomal RNA precursors, located in the nucleolus.
2. RNA Pol II: Responsible for mRNA and long non-coding RNAs; transcribes genes in the nucleoplasm.
3. RNA Pol III: Responsible for small RNAs (tRNA, microRNAs).

RNA Pol II Promoters: Include a TATA box (sequence TATAAA) which is bound by a TATA binding protein.
Transcription factors (TFII) aid in forming a closed complex, similar to sigma factors in prokaryotes.
Specific interactions lead to the advancement to open complex formation and initiation of elongation.

TFII complex is recruited, with TFIIH playing a crucial role in helicase activity, facilitating DNA unwinding.
C-terminal Domain Phosphorylation: Critical for activation and elongation; acts as a 'phosphorylation code' to trigger specific protein recruitment.
Upon adequate RNA synthesis, general transcription factors detach, allowing elongation to commence.

Mediator complex facilitates communication between general transcription factors and enhancer sequences, enhancing RNA Polymerase II activation.

Torpedo Model of Termination: Involves RNA cleavage by endonuclease, followed by degradation of residual RNA by XRN2.
Ensures polymerization completion and prevents unwanted transcription run-ons.

RNA processing is exclusive to eukaryotes; bacterial transcripts lack modifications post-transcription.
Involves capping, polyadenylation, and splicing of mRNA.

Capping Function: Protects RNA from exonuclease degradation; first step during transcription.
Structure of the Cap: A 7-methylguanylate nucleotide linked by a 5’-5’ triphosphate bond to the RNA.
The cap enhances ribosome binding for efficient translation in the cytoplasm.
Capping is initiated by guanylyltransferase upon polymerase C-terminal domain phosphorylation.

Occurs post-transcription at the polyadenylation signal (AAUAAA).
Endonuclease activity cuts the RNA, followed by recruitment of poly A polymerase adding adenosines at the 3' end (200-250 As).
Polyadenylation provides additional stability through the binding of poly A binding protein, despite targeting down-stream RNA degradation by XRN2.

Eukaryotic genes generally contain introns which are removed during splicing.
The remaining exons are spliced together to produce mature mRNA.
Regulatory Function of Introns: Can contain sequences important for gene regulation.
Alternative Splicing: Mechanism by which different protein variants are derived from the same gene via selective exon inclusion.

Creates diversity in proteins, essential for proper cellular function. Dysregulation can cause diseases due to improper protein variants being expressed.
Splicing can involve alternative start and polyadenylation sites, altering the resultant mRNA and protein structure.

Transcription in prokaryotes involves sigma factors binding to promoters leading to RNA polymerase action; termination can occur through rho-independent or rho-dependent mechanisms.
In eukaryotes, transcription is more complex involving general and specific transcription factors, multiple RNA polymerases, capping, and polyadenylation processes to ensure stability and control over gene expression. Rho-dependent termination is replaced by a more intricate torpedo model in eukaryotic cells, promoting efficient RNA processing before translation occurs.

Preparation for the upcoming lessons on RNA processing with a focus on splicing mechanics.