Lecture 6: Eukaryotic transcription – Part 1
Importance of eukaryotic transcription:
• Fundamental process in biology: first step in the central dogma
• Miss-regulation leads to problems, e.g. disease
• Manipulating gene expression used in biotechnology, crop improvement, therapy
• Transcription is a multi-step process that is very tightly regulated: it is complex
The transcription cycle:
Transcription = DNA-templated RNA synthesis, carried out by RNA Polymerases. Three
stages that all RNA polymerases go through: initiation → elongation → termination.
• Initiation: RNAPII binds to the promoter with the help of transcription factors, unwinds DNA, and begins RNA synthesis.
• Elongation: RNAPII moves along the DNA, adding nucleotides to the growing RNA strand while interacting with elongation factors.
• Termination: Transcription ends, RNA is released, and RNAPII disengages from DNA. Termination can occur via different mechanisms
depending on the organism.
How polymerases go through these stages varies. Each matrix-covered DNA segment is a gene coding for rRNA
precursor molecules.
Question: On which side does initiation happen? S or M?
Answer: Initiation begins on the S-side because at the initiation side there will be less RNA-synthesis.
RNA Polymerase:
Bacteria: one RNA Polymerase; eukaryotes: several
polymerases. ~9 bp RNA-DNA hybrid is a major contributor to
the stability of elongating RNA polymerase.
Functions of RNA Polymerase:
• Recognize template DNA bases
• Faithfully add complementary RNA base
• Separate RNA-DNA hybrid
• Separate DNA strands
• Re-anneal DNA strands
RNA polymerization happens 5’ to 3’, antiparallel to the template strand. Polymerase adds to RNA 3’end.
Question: If the 8 nucleotide of DNA template inside the RNAPII active site is 5’- TCCTCTCG -3’. What is the sequence of the RNA 5’ to 3’?
Answer: Use the non-template strand to think of RNA sequence: 5’-CGAGAGGA-3’.
Different types of RNA-polymerases:
• RNA-polymerase I (first peak):
o Alpha-amanitin resistant
o In the nucleolus, where ribosomal RNA is made
o RNA-Pol I transcribes rRNA
• RNA-polymerase II (second peak):
o Alpha-amanitin sensitive → alpha-amanitin causes major RNA loss
o In the nucleoplasm
o RNA-Pol II transcribes mRNA, miRNAs, snRNAs, other non-coding RNAs
• RNA-polymerase III (third peak):
o Alpha-amanitin resistant
o In the nucleoplasm
o RNA-Pol III transcribes tRNAs, 5S rRNA, some other non-coding RNAs
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, Different types of RNA-polymerases:
• Other RNA-polymerases (land plants and some algae):
o RNA-Pol IV transcribes siRNA precursors
o RNA-Pol V transcribes long non-coding RNAs (lncRNAs)
o siRNAs base pairing to lncRNAs cause silencing
• Transcription outside the nucleus:
o Chloroplasts encode and use bacteria-like RNA-Pol
o Mitochondrial DNA is transcribed by nuclear-encoded bacteriophage-like RNA-Pol
Different parts in a transcribing RNA-Pol II structure:
A transcribing RNAPII structure looks like a crab claw and consists of:
• DNA entry and exit channels – where the template DNA enters and exits
• RNA exit channel – where the growing RNA strand emerges
• Active site – where catalysis occurs
• Clamp domain – holds the DNA-RNA hybrid stable
• Carboxyl-terminal domain (CTD) – involved in regulation and RNA processing
RNAPII C-terminal domain:
The C-terminal domain (CTD) of RNAPII consists of repeats of the heptapeptide YSPTSPS. CTD phosphorylation is dynamic
and coordinates transcription with RNA processing.
Phosphorylation of specific residues regulates transcription:
• Ser5 phosphorylation → Promotes initiation and recruitment of capping enzymes (highly expressed in TSS).
• Ser2 phosphorylation → Signals elongation and splicing factor recruitment (highly expressed in PolyA-site).
• Ser7 phosphorylation → Plays a role in snRNA transcription.
CTD is a major interaction surface:
• RNA capping relies on Ser5 phosphorylation
• Ser2ph bound by H3K36 methyltransferase
• Termination relies on Ser2 phosphorylation
Highlights:
• RNA gets made 5’ to 3’, antiparallel from the template strand
• Bacteria have one RNAP, while eukaryotes have 3-5 nuclear RNA polymerases.
o Each polymerase produces specific transcript classes
• All RNA polymerases have a “crab claw” shape core
• Eukaryotic RNA polymerases also have specific subunits
• Repetitive C-terminal domain is not seen in structure, but very important as an interaction surface
Key steps inside RNAPII:
• One DNA base pair melts, the flipped-out DNA base from the template strand is stabilized by bridge helix
• Random rNTPs enter active site. If rNTP is complementary to the flipped-out DNA base, it can stay
• Trigger loop closes in to better position rNTP, reaction occurs
• Translocation happens: RNAPII moves up by one nucleotide
o One more nucleotide of the RNA sticks out of the RNA exit channel
o Base pair reforms between template and non-template DNA strands
rNTP- misincorporation:
• RNAPII error rate is ~5 ∙ 10-6 / bp → 1 mutation per 200k nt
• Average protein-coding gene is 54kb, ~200k mRNAs per cell → ~0.25 mistakes per pre-mRNA, ~50k wrong RNA bases in every cell
• DNA replication errors: ~10-9 bp
• Initial synthesis fidelity: ~10-4 bp
2|Page
Importance of eukaryotic transcription:
• Fundamental process in biology: first step in the central dogma
• Miss-regulation leads to problems, e.g. disease
• Manipulating gene expression used in biotechnology, crop improvement, therapy
• Transcription is a multi-step process that is very tightly regulated: it is complex
The transcription cycle:
Transcription = DNA-templated RNA synthesis, carried out by RNA Polymerases. Three
stages that all RNA polymerases go through: initiation → elongation → termination.
• Initiation: RNAPII binds to the promoter with the help of transcription factors, unwinds DNA, and begins RNA synthesis.
• Elongation: RNAPII moves along the DNA, adding nucleotides to the growing RNA strand while interacting with elongation factors.
• Termination: Transcription ends, RNA is released, and RNAPII disengages from DNA. Termination can occur via different mechanisms
depending on the organism.
How polymerases go through these stages varies. Each matrix-covered DNA segment is a gene coding for rRNA
precursor molecules.
Question: On which side does initiation happen? S or M?
Answer: Initiation begins on the S-side because at the initiation side there will be less RNA-synthesis.
RNA Polymerase:
Bacteria: one RNA Polymerase; eukaryotes: several
polymerases. ~9 bp RNA-DNA hybrid is a major contributor to
the stability of elongating RNA polymerase.
Functions of RNA Polymerase:
• Recognize template DNA bases
• Faithfully add complementary RNA base
• Separate RNA-DNA hybrid
• Separate DNA strands
• Re-anneal DNA strands
RNA polymerization happens 5’ to 3’, antiparallel to the template strand. Polymerase adds to RNA 3’end.
Question: If the 8 nucleotide of DNA template inside the RNAPII active site is 5’- TCCTCTCG -3’. What is the sequence of the RNA 5’ to 3’?
Answer: Use the non-template strand to think of RNA sequence: 5’-CGAGAGGA-3’.
Different types of RNA-polymerases:
• RNA-polymerase I (first peak):
o Alpha-amanitin resistant
o In the nucleolus, where ribosomal RNA is made
o RNA-Pol I transcribes rRNA
• RNA-polymerase II (second peak):
o Alpha-amanitin sensitive → alpha-amanitin causes major RNA loss
o In the nucleoplasm
o RNA-Pol II transcribes mRNA, miRNAs, snRNAs, other non-coding RNAs
• RNA-polymerase III (third peak):
o Alpha-amanitin resistant
o In the nucleoplasm
o RNA-Pol III transcribes tRNAs, 5S rRNA, some other non-coding RNAs
1|Page
, Different types of RNA-polymerases:
• Other RNA-polymerases (land plants and some algae):
o RNA-Pol IV transcribes siRNA precursors
o RNA-Pol V transcribes long non-coding RNAs (lncRNAs)
o siRNAs base pairing to lncRNAs cause silencing
• Transcription outside the nucleus:
o Chloroplasts encode and use bacteria-like RNA-Pol
o Mitochondrial DNA is transcribed by nuclear-encoded bacteriophage-like RNA-Pol
Different parts in a transcribing RNA-Pol II structure:
A transcribing RNAPII structure looks like a crab claw and consists of:
• DNA entry and exit channels – where the template DNA enters and exits
• RNA exit channel – where the growing RNA strand emerges
• Active site – where catalysis occurs
• Clamp domain – holds the DNA-RNA hybrid stable
• Carboxyl-terminal domain (CTD) – involved in regulation and RNA processing
RNAPII C-terminal domain:
The C-terminal domain (CTD) of RNAPII consists of repeats of the heptapeptide YSPTSPS. CTD phosphorylation is dynamic
and coordinates transcription with RNA processing.
Phosphorylation of specific residues regulates transcription:
• Ser5 phosphorylation → Promotes initiation and recruitment of capping enzymes (highly expressed in TSS).
• Ser2 phosphorylation → Signals elongation and splicing factor recruitment (highly expressed in PolyA-site).
• Ser7 phosphorylation → Plays a role in snRNA transcription.
CTD is a major interaction surface:
• RNA capping relies on Ser5 phosphorylation
• Ser2ph bound by H3K36 methyltransferase
• Termination relies on Ser2 phosphorylation
Highlights:
• RNA gets made 5’ to 3’, antiparallel from the template strand
• Bacteria have one RNAP, while eukaryotes have 3-5 nuclear RNA polymerases.
o Each polymerase produces specific transcript classes
• All RNA polymerases have a “crab claw” shape core
• Eukaryotic RNA polymerases also have specific subunits
• Repetitive C-terminal domain is not seen in structure, but very important as an interaction surface
Key steps inside RNAPII:
• One DNA base pair melts, the flipped-out DNA base from the template strand is stabilized by bridge helix
• Random rNTPs enter active site. If rNTP is complementary to the flipped-out DNA base, it can stay
• Trigger loop closes in to better position rNTP, reaction occurs
• Translocation happens: RNAPII moves up by one nucleotide
o One more nucleotide of the RNA sticks out of the RNA exit channel
o Base pair reforms between template and non-template DNA strands
rNTP- misincorporation:
• RNAPII error rate is ~5 ∙ 10-6 / bp → 1 mutation per 200k nt
• Average protein-coding gene is 54kb, ~200k mRNAs per cell → ~0.25 mistakes per pre-mRNA, ~50k wrong RNA bases in every cell
• DNA replication errors: ~10-9 bp
• Initial synthesis fidelity: ~10-4 bp
2|Page
