RNA Synthesis

DNA sequence that RNA polymerase binds to initiate transcription.

What are the two conserved sequences in prokaryotic promoters?

→ −35 sequence and −10 sequence (TATA box/Pribnow box)

What is the function of the −35 sequence in prokaryotic promoters?

→ It helps RNA polymerase recognize and bind to the promoter.

What is the function of the −10 sequence in prokaryotic promoters?

→ It helps in the melting of DNA to allow transcription initiation.

What is the numbering convention for DNA sequences relative to the transcription start site (TSS)?

→ Negative for upstream (e.g., −10, −35), positive for downstream (e.g., +2, +10)

What happens if the −35 or −10 sequence is mutated?

→ RNA polymerase may fail to bind or initiate transcription.

What strand is used as the template for RNA synthesis?

→ The antisense strand (template strand)

What is the polarity of the RNA transcript relative to the coding strand?

→ It has the same polarity and sequence as the coding strand (except uracil replaces thymine).

What type of regulatory elements work in an orientation-dependent manner?

→ Promoters (−35 and −10 elements)

What type of regulatory elements can work in an orientation-independent manner?

→ Enhancers

What is the site where RNA polymerase attaches to begin transcription?

→ Promoter

In which direction is RNA synthesized during transcription?

→ 5' to 3'

What is the function of the 7-methylguanosine cap added to eukaryotic mRNA?

→ Protects against degradation, aids in translation, and assists with mRNA processing

What are the three major steps of transcription?

→ Initiation, elongation, termination

What happens if RNA polymerase encounters a termination signal?

→ Transcription stops

What is the difference between polycistronic and monocistronic mRNA?

→ Polycistronic = multiple proteins; Monocistronic = one protein

What enzyme is responsible for RNA synthesis?

→ RNA polymerase

Where does RNA polymerase bind to start transcription?

→ Promoter

What is the pre-initiation complex (PIC)?

→ The complex formed when RNA polymerase binds to the promoter and unwinds DNA.

In which direction is RNA synthesized?

→ 5' → 3'

What happens during promoter clearance?

→ RNA polymerase moves away from the promoter after synthesizing the first 10 nucleotides.

What is the role of the sigma factor (σ) in prokaryotic transcription?

→ Guides RNA polymerase to the promoter.

What is the function of topoisomerase during transcription?

→ Prevents supercoiling by relieving torsional stress on the DNA.

What is intrinsic termination?

→ Termination triggered by specific sequences in the DNA and RNA.

What is rho-dependent termination?

→ Termination requiring rho (ρ) factor to catch up with RNA polymerase.

What product is formed at the end of transcription?

→ Primary transcript (RNA)

What are the two key elements of a bacterial promoter?

→ -35 Box (TGTTGACA) and -10 Box (TATAAT)

Why is the TATA box easier to unwind than GC-rich sequences?

→ A-T bonds have two hydrogen bonds, while G-C bonds have three hydrogen bonds (stronger).

What is the role of the rho (ρ) factor in transcription termination?

→ Rho binds to the RNA transcript and disrupts the RNA-DNA hybrid, causing termination.

What type of regulatory protein blocks RNA polymerase from binding to the promoter?

→ Repressor

How does an activator increase transcription?

→ It enhances RNA polymerase binding to the promoter.

What is the role of the promoter in eukaryotic transcription?

→ The promoter directs RNA polymerase II to the correct transcription start site and ensures accurate transcription initiation.

What are the key components of a eukaryotic promoter?

→ TATA box, Inr, and DPE.

What is the role of the CAAT box in transcription?

→ It is a proximal upstream element that regulates the frequency of transcription initiation.

How does TFIIH contribute to the basal transcription complex?

→ TFIIH has helicase activity (unwinds DNA) and kinase activity (activates RNA polymerase II).

What is the function of an enhancer?

→ Enhancers increase transcription and can work in any orientation or location.

What is the TATA box and where is it located?

The TATA box is a DNA sequence (consensus: TATAAA) located 25–30 bp upstream of the transcription start site, bound by the TATA-binding protein (TBP), which helps initiate transcription.

What is the function of the Inr (Initiator sequence)?

The Inr spans the transcription start site (-3 to +5) and directs RNA polymerase II to the correct starting point.

What is the DPE (Downstream Promoter Element) and where is it located?

The DPE is a DNA element located ~25 bp downstream of the TSS with the consensus sequence a/gGa/tCGTG; it helps direct transcription.

What are the GC and CAAT boxes?

• The GC box binds Sp1 and enhances transcription.

• The CAAT box binds CTF and regulates transcription frequency.

How do enhancers and silencers function?

• Enhancers increase transcription.

• Silencers decrease transcription.

• They can act far from the gene and are orientation-independent.

What is the role of the AAUAAA signal?

The sequence AAUAAA signals RNA cleavage and polyadenylation, marking the end of transcription.

What is a nucleosome composed of?

→ A nucleosome consists of ~150 bp of DNA wrapped around a histone octamer (2 copies each of H2A, H2B, H3, and H4).

How does histone acetylation affect chromatin structure?

→ Histone acetylation by HAT loosens chromatin, making it more accessible for transcription.

What role do ATP-dependent chromatin remodelers play in transcription?

→ They use ATP to slide, move, or remove nucleosomes, allowing transcription machinery to access DNA.

What is the role of the Pre-Initiation Complex (PIC)?

→ The PIC, composed of RNA polymerase II and general transcription factors, initiates transcription at the promoter.

How can histone methylation affect transcription?

→ Histone methylation by SET can either increase or decrease transcription depending on the specific site modified.

What are the three types of RNA polymerases and their functions in eukaryotic transcription?

• Pol I → Transcribes rRNA

• Pol II → Transcribes mRNA and some small RNAs

• Pol III → Transcribes tRNA and 5S rRNA

What is the function of TFIID in eukaryotic transcription?

• Binds to the TATA box using TBP

• Bends the DNA by ~100° to help position the transcription machinery

How does TFIIH contribute to transcription initiation?

• Unwinds DNA (helicase activity)

• Phosphorylates the tail (CTD) of RNA polymerase II to initiate transcription

What role do coactivators play in transcription?

• Bridge between activator proteins and the transcription complex to regulate transcription rate

What happens when a promoter lacks a TATA box?

• The Inr (initiator) and DPE (downstream promoter element) position the complex

What blocks the promoter from being accessed by Pol II?

→ Nucleosomes wrap around promoter sequences, preventing transcription machinery from binding.

What role does chromatin remodeling play in transcription?

→ Chromatin remodeling complexes like Swi/Snf and p300/CBP displace nucleosomes, exposing the promoter.

How is RNA Pol II activated for transcription?

→ Phosphorylation of the CTD (on Ser and Thr) by kinases like TFIIH activates Pol II.

What are the three classes of transcription factors involved in pol II gene regulation?

→ Pol II and GTFs, coregulators, and DNA-binding activator/repressor proteins.

How does the mediator complex influence transcription?

→ It bridges upstream activators with the Pol II complex and helps regulate transcription.

What is the function of the CTD in RNA polymerase II?

→ The C-terminal domain (CTD) acts as a platform for binding proteins involved in mRNA processing (capping, splicing, 3'-end formation) and is essential for transcription initiation and elongation.

How does phosphorylation of the CTD affect RNA polymerase II activity?

→ Phosphorylation of the CTD activates Pol II and allows transcription to proceed; dephosphorylation reduces activity and can terminate transcription.

What is the mediator complex, and what is its role in transcription?

→ The mediator complex (Med1–Med31) is a multi-protein complex that helps regulate transcription by linking activators to Pol II and assisting with PIC formation.

What is the difference between the stepwise assembly model and the recruitment model of PIC formation?

→ Stepwise model = gradual assembly of the PIC; Recruitment model = preformed complex of Pol II and GTFs is recruited by activators.

What is the role of TFIID in transcription?

→ TFIID consists of TBP and TAFs; TBP binds to the TATA box, and TAFs help recruit and stabilize the transcription complex.

What is the function of the RNA-DNA hybrid during transcription elongation?

→ The RNA-DNA hybrid (~8–9 base pairs) helps stabilize Pol II and ensures proper RNA synthesis.

How does RNA polymerase II proofread the growing RNA strand?

→ Pol II pauses upon detecting an error, uses its nuclease activity to remove the incorrect nucleotide, and then resumes transcription.

What role does P-TEFb play in transcription elongation?

→ P-TEFb phosphorylates Ser2 of the CTD and NELF/DSIF to resume transcription after Pol II pausing.

What is the role of the FACT complex during transcription?

→ FACT complex removes and repositions nucleosomes to allow Pol II to pass through chromatin.

What are the key elongation factors involved in RNA synthesis?

→ TFIIS (proofreading), ELL (increases rate), P-TEFb (resumes transcription), Spt4/Spt5 (increases processivity).

What is the difference between prokaryotic and eukaryotic RNA processing?

→ In prokaryotes, transcription and translation are coupled, with little RNA processing. In eukaryotes, transcription and processing occur in the nucleus before export to the cytoplasm for translation.

What are the key steps in eukaryotic mRNA processing?

→ Capping (5' cap), splicing (removal of introns), and polyadenylation (addition of poly-A tail).

How does splicing occur in eukaryotic cells?

→ The spliceosome (made of snRNPs) removes introns, forms a lariat structure, and joins exons together.

What is the role of U1 and U2 in splicing?

→ U1 binds to the 5' splice site; U2 binds to the branch point, exposing the reactive adenine.

What is alternative splicing and why is it important?

→ Alternative splicing allows a single gene to code for multiple proteins, increasing genetic diversity.

What is the clinical relevance of splicing errors?

→ Errors in splicing can cause diseases like β-thalassemia by altering the reading frame of mRNA.

How is ribosomal RNA (rRNA) processed?

→ rRNA is transcribed as a large precursor (45S) and processed into 28S, 18S, and 5.8S rRNAs in the nucleolus.

What is the role of the 5' cap and poly-A tail?

→ The 5' cap protects mRNA from degradation and helps ribosome recognition; the poly-A tail stabilizes mRNA and regulates its lifespan.

How does alternative promoter utilization regulate gene expression?

→ Different promoters in different tissues lead to tissue-specific gene expression (e.g., glucokinase gene).

What is the function of the SAGA and TREX complexes in RNA processing?

→ SAGA activates transcription and recruits TREX, which links transcription, splicing, and nuclear export.