Lecture 18 Transcription, RNA Processing

What are the key structural and functional differences between prokaryotic and eukaryotic transcription?

Prokaryotic transcription occurs in the cytoplasm, is coupled with translation, and involves fewer regulatory elements. Eukaryotic transcription occurs in the nucleus, is separated from translation, involves RNA Pol II and many transcription factors, and requires RNA processing.

Why are transcription and translation coupled in bacteria but separated in eukaryotes?

Bacteria lack a nucleus, so transcription and translation both occur in the cytoplasm and can happen simultaneously. Eukaryotes have a nucleus, which compartmentalizes transcription.

How does the compartmentalization of transcription and translation benefit eukaryotic cells?

It allows for quality control and processing (e.g., splicing, capping, polyadenylation) before mRNA is

translated, increasing regulation and versatility.

What is the role of RNA Polymerase II, and how does it differ from Pol I and Pol III?

RNA Pol II transcribes mRNA and some snRNAs; Pol I transcribes rRNA, and Pol III transcribes tRNA and 5S rRNA.

What are promoters and what sequence elements are commonly found in eukaryotic promoters?

Promoters are DNA sequences near the transcription start site; they often include a TATA box recognized

by TBP.

What is the function of TFIID, and how does TBP assist in transcription initiation?

TFIID contains TBP, which binds to the TATA box and bends DNA to recruit other transcription factors.

How do general transcription factors assist in assembling the transcription machinery?

They help position RNA Pol II at the promoter, unwind DNA, and initiate transcription.

What role does TFIIH play in promoter melting and CTD phosphorylation?

TFIIH has helicase activity for DNA unwinding and kinase activity to phosphorylate RNA Pol II's CTD.

How do enhancers differ from promoters, and how do they influence gene expression?

Enhancers can be distant from the gene, bind activators/repressors, and interact with the promoter via mediator complexes.

What are the functions of activators and repressors in transcriptional regulation?

Activators enhance transcription by loosening chromatin and aiding initiation; repressors condense

chromatin and block transcription.

What is the role of the Mediator complex in transcription initiation?

It bridges enhancers with the transcription machinery at the promoter.

How does chromatin structure influence transcription?

Open (euchromatin) regions are accessible for transcription; closed (heterochromatin) regions are

transcriptionally silent.

What are the differences between euchromatin and heterochromatin?

Euchromatin is loosely packed and transcriptionally active; heterochromatin is tightly packed and inactive.

How do histone modifications affect gene accessibility?

Acetylation generally activates transcription; methylation can activate or repress depending on the site.

Which enzymes act as 'writers' and 'erasers' for epigenetic marks?

Writers: DNMTs, HATs, HMTs. Erasers: TETs, HDACs, LSDs.

What is the significance of the C-terminal domain (CTD) of RNA Polymerase II?

It coordinates transcription with RNA processing by serving as a scaffold for binding factors.

What do Ser5 and Ser2 phosphorylation indicate during transcription?

Ser5: promoter escape/initiation. Ser2: elongation and RNA processing.

How does CTD phosphorylation coordinate transcription with mRNA processing?

It recruits capping enzymes, splicing factors, and polyadenylation machinery sequentially.

When is the 5' cap added and what is its function?

It is added co-transcriptionally and protects mRNA from degradation, aiding translation.

What is the role of the poly(A) tail and how is it added?

It stabilizes mRNA, aids nuclear export, and is added post-transcriptionally by poly(A) polymerase.

Why are these modifications critical for mRNA stability and translation?

They prevent exonuclease degradation and enhance translation efficiency.

What is the difference between exons and introns?

Exons are coding regions retained in mature mRNA; introns are removed during splicing

What are the main steps involved in spliceosome-mediated splicing?

Recognition of splice sites, lariat formation, and exon ligation.

What is a lariat structure and how is it formed in Group II intron splicing?

The 2' OH of an adenine in the intron attacks the 5' splice site, forming a looped lariat.

How do Group I and Group II introns differ in their splicing mechanisms?

Group I uses a free guanosine; Group II forms a lariat via an internal adenine.

What components make up the spliceosome and what is its function?

snRNAs and proteins; it catalyzes the removal of introns from pre-mRNA.

What is alternative splicing and why is it advantageous?

It allows one gene to produce multiple proteins, increasing functional diversity.

How can alternative splicing affect protein function and cellular response?

It can create proteins with different domains, affecting localization, function, or stability.

What are examples of tissues or processes where alternative splicing plays a crucial role?

Neurons, muscle cells, and during development or stress responses.

Why does the cell invest energy into making complex mRNA modifications and splicing

mechanisms?

To ensure accuracy, flexibility, and regulation of gene expression.

How might defects in splicing or epigenetic regulation lead to disease?

They can cause mis-spliced transcripts or inappropriate gene silencing, contributing to cancer or genetic disorders.

How could you experimentally determine if a gene is being actively transcribed in a eukaryotic

cell?

Use techniques like RT-PCR, RNA-seq, or detect Pol II CTD phosphorylation.

How does transcriptional regulation tie into gene expression in development or stress

responses?

Transcription factors, enhancers, and epigenetic changes allow precise control of gene expression in

response to signals.