DNA Transcription
Similarities and differences between DNA replication and transcription
Similarities
- Addition of nucleotides to 3' end of growing chain
- Direction of growth: 5' - 3'
- Uses DNA template
- Phases: Initiation, Elongation, Termination
Differences
- Transcription does not require a primer (Replication: DNA Polymerase need RNA Primer)
- Not all of the DNA is transcribed (Large part of the genome is never transcribed and made into RNA but replicated)
- Only 1 strand of the DNA is transcribed by RNA polymerase
Requirements of RNA polymerase for transcription / Subunit Structure of RNA polymerase
→ Transcription is the most highly regulated process in the cell b/c of E cost in using up 2 phosphate group for each nucleotide in RNA for making protein
* So the decision starts at the beginning of the process by RNA polymerase
RNA Polymerase
- (NMP)n + NTP → (NMP)n+1 + PPi
- DNA dependent
- Active site has Mg2+ → second Mg2+ is brought in with new nucleotides in the nucleotide addition and released with pyrophosphate
- Subunit composition: α2ββ'σ → holoenzyme (only this enzyme capable of initating transcription)
- Initiates transcription only at promoter region: conserved sequence at -35 and -10 region → recognised by subunit σ
- σ initiates transcription and dissociates → leaves the core enzyme ( α2ββ') which carries out elongation
- Active site = β and β'
Special role of σ in initiation
Initiation
1. Recognition of primer by subunit σ
- promoter region: conserved sequence at -35 and -10 region (bound by RNA Pol but not made into RNA)
- Strong promoters have UP-element: strongly stimulate transcription by providing an additional specific interaction btwn the RNA Pol and DNA
eg) the sequences directing the expression of rRNA
2. Role of subunit σ
- has high affinity for DNA
- reduce core enzyme's affinity to DNA → allows holoenzyme migrate along DNA until promoter is found and bind specifically
- For regulated gene expression, different σ factors enable binding to different promoters
- alpha helix of σ binds to the major groove of the DNA around -10 region and allow DNA melting
→ transition from closed to open complex
→ alpha helix (helix-turn helix motif) is involved in recognition of -10 region that contains aromatic AAs that can interact with bases
on the non-template strand = stabilises the melted DNA
→ favourable binding interaction btwn ssDNA and σ: 2 bases in the non-template strand are flipped out and inserted into the pockets of σ protein
where favourable interactions stabilise the unwound state of the promoter region
Properties of E.coli promoters
- Many different bacterial promoters reveal high degree of variation but all contain related sequences
→ "Consensus Nucleotide Sequence"
→ "average" / the most common nucleotide found at each position in the collection of promoters
→ -35 and -10 sequences
Steps in transcription initiation by RNA polymerase
1. σ initiation
2. Transition from the closed to the open complex → structural changes and opening of the DNA helix
→ Isomerisation: spontaneous conformational change by the 2 bases flipping into the pockets of σthat stabilise the ssDNA and drive melting
- Downstream DNA enters the active site through the downstream DNA channel
- DNA strands separate from position +3 and reforms at -11
- Isomerisation → the pincers at the front of the enzyme pinch down tightly onto the downstream DNA
→ major shift in the σ 1.1 region, which lies within the active center cleft of the holoenzyme blocking the path
3. RNA Polymerase remain stationary and pulls downstream DNA into itself
- "scrunching"
→ DNA downstream is unwound and pulled into the enzyme while RNA Pol remains stationary and bound to the primer
- expose bases for incoming ribonucleotides by H-bonding
4. RNA polymerase escapes from the promoter region and enter elongation
- promoter clearance and the loss of sigma subunit cause conformational change in the holoenzyme to core enzyme = elongation
form of the RNA Polymerase
Similarities and differences between DNA replication and transcription
Similarities
- Addition of nucleotides to 3' end of growing chain
- Direction of growth: 5' - 3'
- Uses DNA template
- Phases: Initiation, Elongation, Termination
Differences
- Transcription does not require a primer (Replication: DNA Polymerase need RNA Primer)
- Not all of the DNA is transcribed (Large part of the genome is never transcribed and made into RNA but replicated)
- Only 1 strand of the DNA is transcribed by RNA polymerase
Requirements of RNA polymerase for transcription / Subunit Structure of RNA polymerase
→ Transcription is the most highly regulated process in the cell b/c of E cost in using up 2 phosphate group for each nucleotide in RNA for making protein
* So the decision starts at the beginning of the process by RNA polymerase
RNA Polymerase
- (NMP)n + NTP → (NMP)n+1 + PPi
- DNA dependent
- Active site has Mg2+ → second Mg2+ is brought in with new nucleotides in the nucleotide addition and released with pyrophosphate
- Subunit composition: α2ββ'σ → holoenzyme (only this enzyme capable of initating transcription)
- Initiates transcription only at promoter region: conserved sequence at -35 and -10 region → recognised by subunit σ
- σ initiates transcription and dissociates → leaves the core enzyme ( α2ββ') which carries out elongation
- Active site = β and β'
Special role of σ in initiation
Initiation
1. Recognition of primer by subunit σ
- promoter region: conserved sequence at -35 and -10 region (bound by RNA Pol but not made into RNA)
- Strong promoters have UP-element: strongly stimulate transcription by providing an additional specific interaction btwn the RNA Pol and DNA
eg) the sequences directing the expression of rRNA
2. Role of subunit σ
- has high affinity for DNA
- reduce core enzyme's affinity to DNA → allows holoenzyme migrate along DNA until promoter is found and bind specifically
- For regulated gene expression, different σ factors enable binding to different promoters
- alpha helix of σ binds to the major groove of the DNA around -10 region and allow DNA melting
→ transition from closed to open complex
→ alpha helix (helix-turn helix motif) is involved in recognition of -10 region that contains aromatic AAs that can interact with bases
on the non-template strand = stabilises the melted DNA
→ favourable binding interaction btwn ssDNA and σ: 2 bases in the non-template strand are flipped out and inserted into the pockets of σ protein
where favourable interactions stabilise the unwound state of the promoter region
Properties of E.coli promoters
- Many different bacterial promoters reveal high degree of variation but all contain related sequences
→ "Consensus Nucleotide Sequence"
→ "average" / the most common nucleotide found at each position in the collection of promoters
→ -35 and -10 sequences
Steps in transcription initiation by RNA polymerase
1. σ initiation
2. Transition from the closed to the open complex → structural changes and opening of the DNA helix
→ Isomerisation: spontaneous conformational change by the 2 bases flipping into the pockets of σthat stabilise the ssDNA and drive melting
- Downstream DNA enters the active site through the downstream DNA channel
- DNA strands separate from position +3 and reforms at -11
- Isomerisation → the pincers at the front of the enzyme pinch down tightly onto the downstream DNA
→ major shift in the σ 1.1 region, which lies within the active center cleft of the holoenzyme blocking the path
3. RNA Polymerase remain stationary and pulls downstream DNA into itself
- "scrunching"
→ DNA downstream is unwound and pulled into the enzyme while RNA Pol remains stationary and bound to the primer
- expose bases for incoming ribonucleotides by H-bonding
4. RNA polymerase escapes from the promoter region and enter elongation
- promoter clearance and the loss of sigma subunit cause conformational change in the holoenzyme to core enzyme = elongation
form of the RNA Polymerase
