2. RNA Synthesis (Transcription)

What is transcription?

Process of synthesizing RNA from a DNA template using complementary base pairing

What is the central dogma?

DNA → RNA → Protein.

What is the structure of prokaryotic RNA polymerase?

Core enzyme = α₂ββ’ω; + σ factor → holoenzyme (required for initiation).

What are the functions of RNA polymerase subunits?

• α (2): assembly + promoter/regulatory interaction

• β: catalytic activity (RNA synthesis)

• β’: DNA binding

• ω: stability

What is the role of the sigma factor?

Recognizes -10 and -35 promoter regions, directs RNA polymerase to start site, then dissociates after initiation (~10 nt).

What does RNA Polymerase I do?

Synthesizes rRNA

What does RNA Polymerase II do?

mRNA + snRNA

What does RNA Polymerase III do?

• tRNA + 5S rRNA

What is mRNA?

• Carries genetic info from DNA

• amino acid Template for protein synthesis

• In eukaryotes → made by RNA polymerase II

Function of tRNA?

• curved croissant

• Transfers amino acids to ribosome to make proteins

• anticodon on tRNA matches/reads the codon on the mRNA

• Synthesized by RNA polymerase III

Function of rRNA?

• Structural + catalytic component of ribosome

• measured in Svedburg units (how fast a particle sediments during centrifugation)

• ~60% rRNA, 40% protein

• Synthesized by RNA pol I (mostly) + III

  

Key prokaryotic promoter elements in prokaryotes?

• -7 to -10 nt (Pribnow/TATAAT)

• -35 box at -35 region (TTGACA)

• upstream promoter (UP) element (A-T rich)….. found in highly expressed genes, bound by a subunit of RNA pol

What factor recognizes prokaryotic promoters?

σ factor

Other regulatory sequences in prokaryotes? to control transcription

• Activator binding site

• Operator (repressor binding site)

Key elements in eukaryotic promoters?

• TATA box (core promoter)

• CAAT box (high transcription genes)

• GC box (↑ transcription, may lack TATA)

• bind transcription factors and recruit RNA polymerase

• 

Where are eukaryotic promoters located?

~25–200 bp upstream of transcription start

Difference between enhancers and silencers?

• Enhancers → ↑ transcription (activators bind)

• Silencers → ↓ transcription (repressors bind)

• Can be far away (upstream/downstream)

What are CpG islands?

• CG-rich promoter regions

• Often unmethylated in active genes

Effect of CpG methylation?

• Represses transcription by proteins that bind to methylated CpG islands

• Blocks transcription machinery

• Seen in cancer (tumor suppressor silencing)

Effects of histone modifications?

• Methylation → heterochromatin (condensed) → ↓ transcription

• Acetylation → euchromatin (relaxed) → ↑ transcription

Coding vs template strand?

• Coding (sense): same sequence as RNA. DNA = RNA.

• non-coding Template (antisense): used for transcription

Steps of transcription?

• Initiation

• Elongation

• Termination

Steps of initiation in prokaryotes?

• RNA pol holoenzyme binds promoter → closed complex

• DNA unwinds → open complex

• Transcription bubble forms

• σ factor released

How does prokaryotic elongation occur?

• RNA pol moves 3’→5’ on DNA

• RNA synthesized 5’→3’

• Bubble ~8–9 nucleotides

Two types of prokaryotic termination?

• Rho-independent

• Rho-dependent

Rho-independent
Q: Mechanism?

• Hairpin loop forms 15-20 nt before end

• U-rich region weakens binding

• RNA dissociates

Rho-dependent
Q: Mechanism?

• Rho binds rut site (rho utilization site)

• Moves along RNA (ATP-dependent)

• Detaches mRNA from polymerase

What is the transcription bubble and why is it important?

• A small unwound region of DNA where transcription occurs

• Size: ~8–9 nt (prokaryotes), ~17 bp (eukaryotes)

• RNA polymerase keeps it open while moving along DNA

• Allows base pairing between template DNA and growing RNA

• DNA re-anneals behind the polymerase

What is required for eukaryotic initiation?

• Transcription factors

• Pre-initiation complex

• RNA pol II recruitment

Key features of elongation in eukaryotes?

• RNA pol II phosphorylation required

• initiation factors shed

• ~17 bp transcription bubble

• Produces pre-mRNA

What does Actinomycin D do?

• Intercalates DNA

• Blocks elongation in proks and euks

• Used in chemotherapy

Function of 5’ cap?

• Stability

• Nuclear export

• Translation initiation

Function of poly(A) tail?

• Stability

• Translation efficiency

• Degradation when shortened in bacteria

What is mRNA splicing?

Removal of introns from pre-mRNA

What is the spliceosome?

• ribonucleoprotein complex: snRNA + proteins (snRNPs)

• Removes introns

What is the difference between introns and exons?

• Introns → non-coding sequences

  • Removed during splicing

  • Not translated into protein

• Exons → coding sequences

  • Kept in mature mRNA

  • Translated into protein

👉 Memory tip:
Exons = Expressed
Introns = In between (cut out)

Major spliceosome components?

U1, U2, U4, U5, U6

>150 proteins

▪ Introns 5’ end splice site GU; 3’ end splice site AG

Key splice site sequences?

• 5’ → GU

• 3’ → AG

Steps of splicing?

• U1 binds 5’ site

• U2 binds branch point

• U4/U5/U6 assemble

• Introns removed

• Exons joined

Why is alternative splicing important?

• 1 gene → multiple proteins

• ~90–95% human genes use it

• ‘3 UTR and ‘5UTR

Role of UTRs?

Control mRNA stability, localization, translation. recognized by miRNA, their associated machinery, and RNA binding proteins

Steps in tRNA processing?

• Remove 5’ leader

• Remove introns

• Add CCA tail

• Base modifications

What is copy number?

Number of transcripts per cell

Does copy number vary?

Yes — depends on gene function and cell needs

Which strand is used for transcription?

Template (antisense) strand

What is the first RNA product in eukaryotes?

Pre-mRNA (contains introns + exons)

Does mature mRNA contain introns?

❌ No

Which element can be far from gene and increases transcription?

Enhancer