Speaker A
Transcription by RNA Polymerase II
Detailed explanation of RNA Polymerase II transcription, covering initiation, elongation, stalling, and termination processes in eukaryotic cells.
Key Takeaways
- Transcription initiation is a multi-step process requiring precise assembly of transcription factors and RNA polymerase II.
- Phosphorylation of the RNA polymerase II CTD regulates RNA processing and transition from initiation to elongation.
- RNA polymerase II can stall and backtrack, but transcription factors can rescue and restart transcription.
- RNA processing, including capping and polyadenylation, is tightly coupled to transcription.
- Termination of transcription involves cleavage of the RNA transcript and addition of a poly A tail before polymerase release.
Summary
- RNA Polymerase II transcribes DNA into RNA using the template strand while the coding strand contains the gene sequence.
- Transcription initiation involves assembly of a complex at the promoter, starting with TF2D binding to the TATA box.
- The transcription initiation complex includes RNA polymerase II and multiple transcription factors (TF2A, TF2B, TF2F, TF2E, TF2H).
- TF2H uses ATP hydrolysis to unwind DNA, forming the transcription bubble and exposing the template strand.
- RNA synthesis begins with abortive initiation, followed by promoter clearance for stable RNA elongation.
- Phosphorylation of the RNA polymerase II CTD by TF2H and P-TEFB is crucial for RNA processing and elongation.
- RNA processing includes addition of a 5' guanosine cap and recruitment of enzymes for transcript maturation.
- During elongation, the transcription bubble moves with RNA polymerase, separating and re-annealing DNA strands.
- RNA polymerase can stall or backtrack; TF2S rescues stalled polymerase by cleaving the RNA to resume transcription.
- Termination occurs after transcribing the polyadenylation signal, leading to RNA cleavage, poly A tail addition, and polymerase dissociation.
Full Transcript — Download SRT & Markdown
Speaker A
To produce a functional RNA molecule, the cell must make an RNA copy of a DNA sequence.
Speaker A
In double-stranded DNA, the strand to be copied is known as the coding strand.
Speaker A
The other strand, which contains the complementary base sequence, is the template strand that will be used to form the RNA transcript.
Speaker A
So what are the key features of the coding strand?
Speaker A
The start of the coding sequence is known as the transcription start site.
Speaker A
The five prime side of this site is the promoter region. The promoter region contains particular DNA sites known as core promoter elements.
Speaker A
These are important in the assembly of the complex of proteins that are needed to initiate transcription.
Speaker A
This complex is called the transcription initiation complex and contains the RNA polymerase II enzyme along with additional proteins or protein complexes known as transcription factors.
Speaker A
Transcription factors help RNA polymerase to locate the promoter and initiate transcription.
Speaker A
Assembly of the Initiation Complex
Speaker A
We'll now consider how the initiation complex assembles.
Speaker A
The first step in the assembly of the transcription initiation complex
Speaker A
is the binding of the transcription factor TF2D to the TATA box,
Speaker A
which is one of the core DNA promoter elements.
Speaker A
TF2D is a complex of proteins and contains the key subunit that binds the TATA box.
Speaker A
This key subunit is known as TATA binding protein or TBP.
Speaker A
The TBP subunit induces profound bending of the DNA.
Speaker A
Binding of TF2D is followed by the binding of TF2A and TF2B.
Speaker A
TF2B binds to the core promoter element called the BRE as well as contacting TF2D.
Speaker A
In the next step, the RNA polymerase II core enzyme is recruited together with TF2F.
Speaker A
Finally, TF2E and TF2H are recruited to form the complete transcription initiation complex.
Speaker A
This is the closed complex, meaning that the DNA is still double-stranded and the template strand has not yet been exposed.
Speaker A
Transcription begins with the separation of the two DNA strands to form the open complex.
Speaker A
This separation depends on TF2H, which uses energy from ATP hydrolysis to unwind the DNA and promote strand opening.
Speaker A
The short stretch of DNA with unpaired strands is called a transcription bubble.
Speaker A
The opening of the transcription bubble exposes the template strand, which will be used to form a complementary RNA strand.
Speaker A
Free ribonucleotides triphosphates enter through the funnel region of the RNA polymerase
Speaker A
and form base pairing interactions with the template strand.
Speaker A
RNA polymerase catalyzes the addition of nucleotides to the three prime hydroxyl of the last base in the growing chain.
Speaker A
This means that the RNA grows in the five prime to three prime direction.
Speaker A
Transcription initiation does not go smoothly.
Speaker A
RNA polymerase typically will start to synthesize the RNA, but the RNA product will diffuse away after it has reached a length of just a few base pairs.
Speaker A
The whole process of synthesis then starts again.
Speaker A
This process is known as abortive initiation.
Speaker A
After multiple rounds of abortive initiation, RNA polymerase breaks free of the promoter
Speaker A
and continues to synthesize the RNA in a more persistent way.
Speaker A
This phase is called promoter clearance.
Speaker A
RNA polymerase now forms a stable complex with the DNA and continues to synthesize the RNA.
Speaker A
At the same time, TF2H phosphorylates the C-terminal domain of the large subunits of RNA polymerase II.
Speaker A
This domain is called CTD.
Speaker A
This phosphorylation of CTD is key to the processing of the RNA to produce the mature transcript, and this is what happens next.
Speaker A
RNA Processing
Speaker A
During the RNA processing step, a complex of RNA processing enzymes called P-TEFB
Speaker A
binds to the phosphorylated CTD.
Speaker A
As the five prime end of the growing RNA comes out of the exit channel of RNA polymerase,
Speaker A
P-TEFB covalently attaches a guanosine cap to the five prime end of the RNA.
Speaker A
P-TEFB then attaches additional phosphate groups to the CTD, which causes transcription elongation, which is paused, to resume.
Speaker A
The more highly phosphorylated CTD will also recruit additional RNA processing enzymes that will be needed to complete processing of the transcript at the end of the transcription cycle.
Speaker A
Elongation
Speaker A
RNA polymerase has now transitioned into the elongation phase.
Speaker A
During this phase, the growing RNA strand comes out of the RNA polymerase exit channel as it continues to elongate.
Speaker A
Transcription bubble moves along with the RNA polymerase.
Speaker A
DNA base pairs are separated ahead of the transcribing polymerase to expose more of the template strand.
Speaker A
More bases in the wake of the polymerase re-anneal.
Speaker A
In this way, RNA polymerase can continue until it has transcribed the complete RNA.
Speaker A
Polymerase Stalling
Speaker A
RNA polymerase doesn't always make it to the end of the coding sequence in one smooth journey.
Speaker A
Instead, RNA polymerase can sometimes stall before reaching the end of the transcribed region.
Speaker A
The polymerase can be restarted by the binding of elongation factors.
Speaker A
But sometimes the RNA polymerase starts to backtrack.
Speaker A
This means that the polymerase slides backwards along with the transcription bubble.
Speaker A
This causes the three prime end of the growing transcript to become unpaired and to come out through the funnel region of the RNA polymerase.
Speaker A
The transcription factor TF2S can rescue the stalled polymerase by binding in the funnel region and promoting cleavage of the three prime end of the RNA that is unpaired.
Speaker A
This exposes a new three prime hydroxyl in the RNA that is correctly positioned in the active site.
Speaker A
Transcription can now resume as before.
Speaker A
Termination of Transcription
Speaker A
Transcription continues until RNA polymerase II reaches a sequence in the DNA
Speaker A
called a polyadenylation signal.
Speaker A
After RNA polymerase has transcribed through this signal, sequences in the RNA transcript associate with the RNA processing proteins
Speaker A
that are bound to the polymerase CTD.
Speaker A
These proteins cleave the RNA transcript and add a sequence of adenine nucleotides to the three prime end of the transcript.
Speaker A
This run of adenines is called a poly A tail.
Speaker A
Once the poly A tail is added, RNA polymerase dissociates from the DNA and the mature RNA is released.
Speaker A
At this point, transcription is terminated and an RNA transcript has been successfully produced.
Topics:RNA Polymerase IItranscription initiationtranscription elongationtranscription terminationtranscription factorsRNA processingCTD phosphorylationabortive initiationpromoter clearancepolyadenylation
