Chapter 13: DNA Transcription

State the central dogma of molecular biology. 

• DNA → RNA → Protein; genetic information is stored in DNA, transcribed into RNA, and translated into proteins.

What other gene products exist besides protein? 

•  mRNA, tRNA, rRNA

What DNA elements/sequences make up a eukaryotic gene? How is that DNA information related to the structure of an mRNA?

• Promoter: Core promoter (including TATA box), proximal promoter elements.

• Enhancers and silencers: Cis-regulatory sequences that increase or decrease transcription.

• Exons: Coding sequences that will appear in mature mRNA.

• Introns: Noncoding sequences spliced out during mRNA processing.

Relation to mRNA:

• Exons correspond to sequences in mature mRNA.

• Introns are removed via splicing.

• Regulatory sequences (promoters, enhancers) control when/where mRNA is produced but are not part of mature mRNA.

What is the difference between the coding strand and template strand? If given either of those sequences, be able to predict the sequence of a transcript with directionality. 

• Template strand: DNA strand read by RNA polymerase; complementary to mRNA.

• Coding strand: DNA strand identical to mRNA (except T → U).

• Prediction:

  • If given template strand: transcribe complementary RNA (5′→3′).

  • If given coding strand: replace T with U to get mRNA.

Which strand does RNA polymerase ‘read’ and in what direction? 

• Reads the template strand in the 3′ → 5′ direction.

In what direction does RNA polymerase synthesize the transcript?

• Synthesizes RNA 5′ → 3′.

List the four major steps in transcription.

• Initiation: RNAP binds promoter (with sigma factor in prokaryotes or GTFs in eukaryotes). DNA unwinds.

• Promoter clearance / abortive initiation: Short transcripts may be released until stable elongation is established.

• Elongation: RNAP adds NTPs complementary to the template strand.

• Termination: RNAP dissociates; in eukaryotes, involves cleavage at poly-A signal.

In prokaryotes, how does RNA polymerase find the TSS? 

• Sigma (σ) subunit of RNAP binds –10 (Pribnow box) and –35 sequences of the promoter.

What is the difference between the core and holoenzyme? 

• Core enzyme: Catalyzes RNA synthesis, cannot initiate at promoter.

• Holoenzyme: Core enzyme + sigma factor; can recognize promoter and initiate transcription.

What is the difference between intrinsic and rho-dependent termination? 

• Intrinsic: GC-rich hairpin + poly-U sequence causes RNAP to fall off.

• Rho-dependent: Rho protein binds rut site → moves along RNA → causes RNAP dissociation.
  

What are the differences between prokaryotic and eukaryotic transcription?

Feature	Prokaryotes	Eukaryotes
Location	Cytoplasm	Nucleus
RNA polymerase	Single type	RNAP I, II, III
Initiation	Sigma factor binds promoter	GTFs + promoters + enhancers/silencers
Chromatin	Not present	Chromatin remodeling required
Termination	Hairpin/poly-U or Rho	Cleavage at polyadenylation signal
mRNA processing	None	5′ cap, splicing, 3′ poly-A tail
RNA type	Polycistronic	Monocistronic (usually)

What is meant by a polycistronic versus a monocistronic RNA? 

• Polycstronic: One RNA encodes multiple proteins (prokaryotes)

• Monocistronic: One RNA encodes a single protein (eukaryotes).

What kinds of RNA do eukaryotic RNA polymerase I, II, and III transcribe? 

Polymerase	RNA Product
I	rRNA
II	mRNA, snRNA, miRNA, lncRNA
III	tRNA, 5S rRNA

What is the role of the general transcription factors? What do they bind to? 

• Required for RNAP II binding and initiation.

• Bind to core promoter elements (e.g., TATA box).

What makes up the PIC? (pre-initiation complex)

• Components: RNAP II + general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH).

• Forms at core promoter before elongation.

What is the order of mRNA maturation events? 

1. 5′ capping (shortly after transcription begins).

2. Splicing of introns → exons joined

3. 3′ polyadenylation.

What is added to the 5’ end of the mRNA? What purpose does it serve? When does this happen? What kind of linkage connects it to the mRNA? 

• What is added: 7-methylguanosine.

• Purpose: Protects from nucleases, aids nuclear export, required for translation.

• Timing: Added shortly after transcription starts.

• Linkage: Unique 5′–5′ triphosphate bond.

What is spliced in the splicing of mRNA? Where are the three splice sites of an mRNA intron located? 

• What is spliced: Introns are removed; exons ligated.

• Three splice sites:

  • 5′ splice donor (GU)

  • 3′ splice acceptor (AG)

  • Branch point adenine (A) in intron

What complex catalyzes splicing? What are the subunits of this complex called? What are they made of? How does the snRNA of these subunits recognize the splice sites? What would happen if the splice sites were mutated? 

• Catalyst: Spliceosome (not autocatalytic for nuclear pre-mRNAs).

• Subunits: snRNPs (U1, U2, U4, U5, U6).

• Composition: snRNA + proteins.

• Recognition: snRNA base-pairs with intron splice site sequences

• Mutation effect: Improper splicing → exon skipping, intron retention, nonfunctional protein.

How is polyadenylation performed as a step in transcription termination? What end of the mRNA is the polyA tail added to? What adds the polyA tail? What is the purpose of the polyA tail?

• Step in transcription termination: RNAP II transcribes past AAUAAA poly-A signal → transcript is cleaved → poly-A tail added.

• End of mRNA: 3′ end.

• Enzyme: Poly-A polymerase.

• Purpose: Stabilizes mRNA, aids nuclear export, facilitates translation initiation.