o The exit of the RNA transcript is sensed by the subunits, which trigger a conformational change of
the clamp, forcing it shut
o The clamp then closes over the transcript, which increases processivity.
o This process occurs between abortive initiation and processive elongation.
- Though they have conditional genomes, their structures have been conserved throughout evolution.
- They also have a strong influence on interactions with general TFs.
o RNAPII cannot bind directly to the promoter DNA, and required general TFs.
o Some TFs make direct contact with the polymerase ‘dock’ region.
o Rpb 4 and 7 extend the dock region, allowing for better binding to the TFs.
- It can likely also bind directly to nascent RNA and direct it towards the CTD for post-transcriptional
modifications.
Eukaryotic Class II Promoters
Eukaryotic Promoters
- Along the genome, there are genes that act as regulators of the activity and expression of other regions of
the genome
- The promoter is such a region, and the first elements that controls transcription
- Since there are three RNA polymerases with different functions and conformations, one can expect that
each one recognizes different promoters.
- Their activity occurs at the pre-initiation steps of transcriptions.
o the promoter is not recognized by RNA polymerases.
o the recognition of the first nucleotide to be transcribed (the TSS) is made by general transcription
factors (TFs)
o the assembly of the polymerases and the transcription apparatus occurs after
- the core promoter can contain any combination of these elements:
o the TATA box: centered at approx. -25, with a consensus sequence TATA(A/T) AA(A/T)
▪ the distance between the TATA box and the transcription initiation sites remains constants
in all organisms, but its function can vary (can act simply as a locator, or can impair promoter
function)
▪ the first 4 nucleotides have >90% frequency
▪ all consensus sequences are found on the coding strand.
o the TFIIB recognition element: lies upstream of the TATA box, with consensus sequence (G/C)
(G/C)(G/A)CGCC
o the initiator (Inr): centered on the TSS (+1), with a consensus sequence YYAN(T/A)YYY (Y =
pyrimidine, C or T)
o the downstream promoter element (DPE)
o the upstream promoter element (UPE)
- - general eukaryotic promoters are the CpG islands, or one made up of several elements (UPE, TATA, Inr,
DPE)
o More than 60% are CpG islands.
o There is no unique TSS in CpG islands (transcription can start at multiple sites), but there is one
unique conserved TSS in TATA-style promoters
- class II promoters recognize RNAPII.
- there are 2 general types: the core promoter and the
proximal promoter
o the core promoter attracts general TFs and
RNAPII at a basal level, and sets the TSS and
direction of transcription; found within approx.
37bp of the TSS, on either side
o the proximal promoter helps attract general TFs
and RNAPII and includes promoter elements
, that can extend up to 250bps upstream of the TSS
- eukaryotic promoters often have a proximal promoter and an upstream promoter.
- enhancers and silencers are found far away from the TSS
- they are associated with cis acting DNA elements that are not strictly part of the promoter, but strongly
influence transcription
o they are asymmetric: they function whether they are upstream, downstream, or inversed (no
connection between activity and orientation)
o enhancers stimulate transcription by increasing the recruitment of RNAPII.
▪ they act through proteins that bind to them (activators), triggering the formation of a
preinitiation complex
▪ often found upstream of the promoters they control.
o silencers repress transcription, usually by controlling chromatin structure.
- the initiator and the TATA box constitute the core promoter of most essential, non-specific genes.
- many genes have more than one activator binding site, so they can respond to multiple stimuli.
- enhancers that interact with many activators allow for a very precise control of gene expression.
o also, because multiple enhancer elements are known to act cooperatively.
Primer Extension
- a method to map the 5’ end of a transcript to the exact nucleotide.
- process:
1. transcription occurs in vivo in a vector cell.
2. one harvests the cellular RNA containing the target transcript.
3. labelled primers are hybridized approx. 18 nts away from the target
RNA sequence (the primer is complementary to the RNA strand, so has
to be prepared in advance)
o because it is labelled, the strand can be isolated from the rest
4. reverse transcriptase is used to extend the primer, making a DNA copy
of the RNA sequence
5. the hybrid is denatured, and sequencing technologies can be applied
- the intensity of the signal obtained by this method is a measure of the
concentration of the transcript in the cell (gives information on level of
expression)
SV40 Early Gene Promoter
- sv40 is a virus that triggers cell transformation in monkeys.
- it only has 1 control regions and an early promoter (for prematurely transcribed genes)
- it does not contain an initiator, only a GC box as an upstream element and the TATA box
- it was used to discover the first enhancers flanking the sv40 early gene, because it contains a 72bp repeat
o deletions of this region caused depressed transcription, suggesting that it is an enhancer
▪ it’s not a promoter, because it was
orientation and position independent
(could work even if it was moved or
inverted)
Upstream Elements
- In general terms, the proximal promoter determines the initiation start site, and enhance transcription
- The upstream promoters are made up of many elements, with the most common being the CAT box and the
GC boxes (both motifs that are GC rich)
o Each consensus sequence is bound by a specific protein.
- GC boxes are found in a variety of promoters, mainly upstream
of the TATA box, and are activated when bound to TF Sp1
the clamp, forcing it shut
o The clamp then closes over the transcript, which increases processivity.
o This process occurs between abortive initiation and processive elongation.
- Though they have conditional genomes, their structures have been conserved throughout evolution.
- They also have a strong influence on interactions with general TFs.
o RNAPII cannot bind directly to the promoter DNA, and required general TFs.
o Some TFs make direct contact with the polymerase ‘dock’ region.
o Rpb 4 and 7 extend the dock region, allowing for better binding to the TFs.
- It can likely also bind directly to nascent RNA and direct it towards the CTD for post-transcriptional
modifications.
Eukaryotic Class II Promoters
Eukaryotic Promoters
- Along the genome, there are genes that act as regulators of the activity and expression of other regions of
the genome
- The promoter is such a region, and the first elements that controls transcription
- Since there are three RNA polymerases with different functions and conformations, one can expect that
each one recognizes different promoters.
- Their activity occurs at the pre-initiation steps of transcriptions.
o the promoter is not recognized by RNA polymerases.
o the recognition of the first nucleotide to be transcribed (the TSS) is made by general transcription
factors (TFs)
o the assembly of the polymerases and the transcription apparatus occurs after
- the core promoter can contain any combination of these elements:
o the TATA box: centered at approx. -25, with a consensus sequence TATA(A/T) AA(A/T)
▪ the distance between the TATA box and the transcription initiation sites remains constants
in all organisms, but its function can vary (can act simply as a locator, or can impair promoter
function)
▪ the first 4 nucleotides have >90% frequency
▪ all consensus sequences are found on the coding strand.
o the TFIIB recognition element: lies upstream of the TATA box, with consensus sequence (G/C)
(G/C)(G/A)CGCC
o the initiator (Inr): centered on the TSS (+1), with a consensus sequence YYAN(T/A)YYY (Y =
pyrimidine, C or T)
o the downstream promoter element (DPE)
o the upstream promoter element (UPE)
- - general eukaryotic promoters are the CpG islands, or one made up of several elements (UPE, TATA, Inr,
DPE)
o More than 60% are CpG islands.
o There is no unique TSS in CpG islands (transcription can start at multiple sites), but there is one
unique conserved TSS in TATA-style promoters
- class II promoters recognize RNAPII.
- there are 2 general types: the core promoter and the
proximal promoter
o the core promoter attracts general TFs and
RNAPII at a basal level, and sets the TSS and
direction of transcription; found within approx.
37bp of the TSS, on either side
o the proximal promoter helps attract general TFs
and RNAPII and includes promoter elements
, that can extend up to 250bps upstream of the TSS
- eukaryotic promoters often have a proximal promoter and an upstream promoter.
- enhancers and silencers are found far away from the TSS
- they are associated with cis acting DNA elements that are not strictly part of the promoter, but strongly
influence transcription
o they are asymmetric: they function whether they are upstream, downstream, or inversed (no
connection between activity and orientation)
o enhancers stimulate transcription by increasing the recruitment of RNAPII.
▪ they act through proteins that bind to them (activators), triggering the formation of a
preinitiation complex
▪ often found upstream of the promoters they control.
o silencers repress transcription, usually by controlling chromatin structure.
- the initiator and the TATA box constitute the core promoter of most essential, non-specific genes.
- many genes have more than one activator binding site, so they can respond to multiple stimuli.
- enhancers that interact with many activators allow for a very precise control of gene expression.
o also, because multiple enhancer elements are known to act cooperatively.
Primer Extension
- a method to map the 5’ end of a transcript to the exact nucleotide.
- process:
1. transcription occurs in vivo in a vector cell.
2. one harvests the cellular RNA containing the target transcript.
3. labelled primers are hybridized approx. 18 nts away from the target
RNA sequence (the primer is complementary to the RNA strand, so has
to be prepared in advance)
o because it is labelled, the strand can be isolated from the rest
4. reverse transcriptase is used to extend the primer, making a DNA copy
of the RNA sequence
5. the hybrid is denatured, and sequencing technologies can be applied
- the intensity of the signal obtained by this method is a measure of the
concentration of the transcript in the cell (gives information on level of
expression)
SV40 Early Gene Promoter
- sv40 is a virus that triggers cell transformation in monkeys.
- it only has 1 control regions and an early promoter (for prematurely transcribed genes)
- it does not contain an initiator, only a GC box as an upstream element and the TATA box
- it was used to discover the first enhancers flanking the sv40 early gene, because it contains a 72bp repeat
o deletions of this region caused depressed transcription, suggesting that it is an enhancer
▪ it’s not a promoter, because it was
orientation and position independent
(could work even if it was moved or
inverted)
Upstream Elements
- In general terms, the proximal promoter determines the initiation start site, and enhance transcription
- The upstream promoters are made up of many elements, with the most common being the CAT box and the
GC boxes (both motifs that are GC rich)
o Each consensus sequence is bound by a specific protein.
- GC boxes are found in a variety of promoters, mainly upstream
of the TATA box, and are activated when bound to TF Sp1
