Epigenetic Regulation of Gene Expression
- Definition: Epigenetics focuses on the mechanisms of transmission of offspring of hereditary traits
independent from genomic sequences
- DNA methylation, post-transcriptional modification of histones, nucleosome remodelers
- Phenotypes are determined by genes and epigenetic regulation.
o Genomic transcripts determine the phenotype.
o The high number of transcripts, the diversity of the proteome and the post-synthesis modification of
proteins increase the complexity and specificity of regulatory mechanisms
RNA Polymerase II
Catalytic Reaction by RNA Polymerase
- The enzymes catalyze the addition of a nucleotide to the 3’ end of the transcript to extend it.
o All nucleic acids in biological systems are synthesized 5’ to 3’
- The product of the reaction will be a transcript with n+1 nucleotides, and a pyrophosphate group (2
phosphates linked together)
- This reaction is catalyzed only when the incoming nucleotide anneals with the base of the template (RNA
polymerase will catalyze the reaction only when the nucleotide is correctly paired)
o The enzymes catalyze the nucleophilic attack of the OH on the 3’ phosphate (α phosphate)
- Magnesium helps compensate for the negative chares of the incoming nucleotide.
- The most important cofactor is the template, because without it the nucleotide cannot stabilize and bind
- The sequence of the transcript is complementary to the template strand.
- The process is very processive.
- The central role of RNA polymerase is to unwind the double helix, polymerase the RNA, and proofread the
transcript
- RNA polymerase II (RNAPII) assembles into larger initiation and elongation complexes, capable of promoter
recognition and response to regulatory signals
o They must bind to elongate and stop transcription.
Transcription Start Site (TSS)
- Synthesis always starts at 3.’
- The last 9-10 bases of the transcript are annealed to the template.
- This hybrid structure should be destroyed (only temporary)
- Pol II destroys the duplex and separates the transcript from the template
- The template is then reannealed to the coding strand when it exits the transcription site
- The TSS is at the beginning of the transcript (+1)
Promoter Classes
- In eukaryotes there are 2 general classes of promoters
- 60-70% of Pol II promoters are CpG islands, regions of the genome that are rich in CpG codons.
- CpG regions are more likely to be methylated.
o Methylation regulatory mechanisms act at Cp regions (a cytosine before a guanine), so they act as
regulatory regions, and are thus underrepresented in the genome
o CpG islands are 300-500 bps long.
, o The CpG island promoters are characterized by multiple TSSs.
- The TATA promoters (20-30%) are characterized by a single TSS
- There can be other upstream and downstream elements.
- The initiation site is labelled as Inr + 1.
- Eukaryotic RNA polymerases cannot recognize and bind directly to the promoters.
o This is not a weakness, but an additional regulatory mechanism; if they could bind autonomously
with high affinity, they would be difficult to regulate
Transcription
- Transcription can be divided into 3 steps
1. Initiation: polymerase binds to the promoter
2. Elongation: the transcription bubble forms, and the template
starts to form
3. Termination: disassembly of the transcription factors and
bubble, and the release of the RNA transcript
- All these steps provide a way of regulating gene expression (most
processes act at pre-initiation and initiation, so no nucleotides and energy are wasted)
Eukaryotic RNA Polymerases
- Transcription is specific in the sense that each polymerase has a specific role.
- Polymerase I: 28S, 18S and 6.8S of the large rRNA regions
o Vast majority of RNA is rRNA, so pol I is very processive
o Concentrated in the nucleolus (the areas where rDNA chromatin is organized)
o Only 1 promoter; least regulated
- Polymerases II and III are speared across the nucleus, and not specified to a specific region.
- Polymerase III: tRNA and 5S rRNA
- Polymerase II: small nuclear RNA (snRNA) and non-coding RNA
o Has many promoter regions which vary in activation, depending on which genes are expressed
o It has many more regulatory mechanisms.
Α-amanitin
- A highly toxic substance that was found to have different effects
on the 3 polymerases
o At low concentrations it inhibits Pol II completely while
having no effect on Pol I and III
o At 1000-fold concentrations, the toxin also inhibits Pol III
for most eukaryotes
o Pol I is completely resistant to it.
RNA Polymerase II Subunits
- RNA Pol II is a protein complex made of 12 subunits.
- Under electrophoresis, the subunits are separated based on molecular weight (the heavier
the protein, the less it travels through the gel)
o The enzyme has multiple subunits which can be present in multiple copies; the
more copies there are, the larger and darker the band
- Radioactive labelling is also used to determine the subunit’s molecular composition and
regulatory mechanisms
o Radioactive sulfur is used to identify methionine and cysteine amino acids; most
abundant in Rpb1 and Rpb2
o radioactive phosphate is used to determine if some subunits are phosphorylated
(the main method of protein function regulation)
▪ the presence of the stain is indicative that the subunit is regulated.
- Definition: Epigenetics focuses on the mechanisms of transmission of offspring of hereditary traits
independent from genomic sequences
- DNA methylation, post-transcriptional modification of histones, nucleosome remodelers
- Phenotypes are determined by genes and epigenetic regulation.
o Genomic transcripts determine the phenotype.
o The high number of transcripts, the diversity of the proteome and the post-synthesis modification of
proteins increase the complexity and specificity of regulatory mechanisms
RNA Polymerase II
Catalytic Reaction by RNA Polymerase
- The enzymes catalyze the addition of a nucleotide to the 3’ end of the transcript to extend it.
o All nucleic acids in biological systems are synthesized 5’ to 3’
- The product of the reaction will be a transcript with n+1 nucleotides, and a pyrophosphate group (2
phosphates linked together)
- This reaction is catalyzed only when the incoming nucleotide anneals with the base of the template (RNA
polymerase will catalyze the reaction only when the nucleotide is correctly paired)
o The enzymes catalyze the nucleophilic attack of the OH on the 3’ phosphate (α phosphate)
- Magnesium helps compensate for the negative chares of the incoming nucleotide.
- The most important cofactor is the template, because without it the nucleotide cannot stabilize and bind
- The sequence of the transcript is complementary to the template strand.
- The process is very processive.
- The central role of RNA polymerase is to unwind the double helix, polymerase the RNA, and proofread the
transcript
- RNA polymerase II (RNAPII) assembles into larger initiation and elongation complexes, capable of promoter
recognition and response to regulatory signals
o They must bind to elongate and stop transcription.
Transcription Start Site (TSS)
- Synthesis always starts at 3.’
- The last 9-10 bases of the transcript are annealed to the template.
- This hybrid structure should be destroyed (only temporary)
- Pol II destroys the duplex and separates the transcript from the template
- The template is then reannealed to the coding strand when it exits the transcription site
- The TSS is at the beginning of the transcript (+1)
Promoter Classes
- In eukaryotes there are 2 general classes of promoters
- 60-70% of Pol II promoters are CpG islands, regions of the genome that are rich in CpG codons.
- CpG regions are more likely to be methylated.
o Methylation regulatory mechanisms act at Cp regions (a cytosine before a guanine), so they act as
regulatory regions, and are thus underrepresented in the genome
o CpG islands are 300-500 bps long.
, o The CpG island promoters are characterized by multiple TSSs.
- The TATA promoters (20-30%) are characterized by a single TSS
- There can be other upstream and downstream elements.
- The initiation site is labelled as Inr + 1.
- Eukaryotic RNA polymerases cannot recognize and bind directly to the promoters.
o This is not a weakness, but an additional regulatory mechanism; if they could bind autonomously
with high affinity, they would be difficult to regulate
Transcription
- Transcription can be divided into 3 steps
1. Initiation: polymerase binds to the promoter
2. Elongation: the transcription bubble forms, and the template
starts to form
3. Termination: disassembly of the transcription factors and
bubble, and the release of the RNA transcript
- All these steps provide a way of regulating gene expression (most
processes act at pre-initiation and initiation, so no nucleotides and energy are wasted)
Eukaryotic RNA Polymerases
- Transcription is specific in the sense that each polymerase has a specific role.
- Polymerase I: 28S, 18S and 6.8S of the large rRNA regions
o Vast majority of RNA is rRNA, so pol I is very processive
o Concentrated in the nucleolus (the areas where rDNA chromatin is organized)
o Only 1 promoter; least regulated
- Polymerases II and III are speared across the nucleus, and not specified to a specific region.
- Polymerase III: tRNA and 5S rRNA
- Polymerase II: small nuclear RNA (snRNA) and non-coding RNA
o Has many promoter regions which vary in activation, depending on which genes are expressed
o It has many more regulatory mechanisms.
Α-amanitin
- A highly toxic substance that was found to have different effects
on the 3 polymerases
o At low concentrations it inhibits Pol II completely while
having no effect on Pol I and III
o At 1000-fold concentrations, the toxin also inhibits Pol III
for most eukaryotes
o Pol I is completely resistant to it.
RNA Polymerase II Subunits
- RNA Pol II is a protein complex made of 12 subunits.
- Under electrophoresis, the subunits are separated based on molecular weight (the heavier
the protein, the less it travels through the gel)
o The enzyme has multiple subunits which can be present in multiple copies; the
more copies there are, the larger and darker the band
- Radioactive labelling is also used to determine the subunit’s molecular composition and
regulatory mechanisms
o Radioactive sulfur is used to identify methionine and cysteine amino acids; most
abundant in Rpb1 and Rpb2
o radioactive phosphate is used to determine if some subunits are phosphorylated
(the main method of protein function regulation)
▪ the presence of the stain is indicative that the subunit is regulated.
