Transcription

Transcription by RNA Polymerase II

To produce a functional RNA molecule, the cell must make an RNA copy of a DNA sequence.

In double-stranded DNA, the strand to be copied is known as the coding strand.

The other strand, which contains the complementary base sequence, is the template strand that will be used to form the RNA transcript.

So what are the key features of the coding strand?

The start of the coding sequence is known as the transcription start site.

The five prime side of this site is the promoter region. The promoter region contains particular DNA sites known as core promoter elements.

These are important in the assembly of the complex of proteins that are needed to initiate transcription.

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.

Transcription factors help RNA polymerase to locate the promoter and initiate transcription.

Assembly of the Initiation Complex

We'll now consider how the initiation complex assembles.

The first step in the assembly of the transcription initiation complex

is the binding of the transcription factor TF2D to the TATA box,

which is one of the core DNA promoter elements.

TF2D is a complex of proteins and contains the key subunit that binds the TATA box.

This key subunit is known as TATA binding protein or TBP.

The TBP subunit induces profound bending of the DNA.

Binding of TF2D is followed by the binding of TF2A and TF2B.

TF2B binds to the core promoter element called the BRE as well as contacting TF2D.

In the next step, the RNA polymerase II core enzyme is recruited together with TF2F.

Finally, TF2E and TF2H are recruited to form the complete transcription initiation complex.

This is the closed complex, meaning that the DNA is still double-stranded and the template strand has not yet been exposed.

Transcription begins with the separation of the two DNA strands to form the open complex.

This separation depends on TF2H, which uses energy from ATP hydrolysis to unwind the DNA and promote strand opening.

The short stretch of DNA with unpaired strands is called a transcription bubble.

The opening of the transcription bubble exposes the template strand, which will be used to form a complementary RNA strand.

Free ribonucleotides triphosphates enter through the funnel region of the RNA polymerase

and form base pairing interactions with the template strand.

RNA polymerase catalyzes the addition of nucleotides to the three prime hydroxyl of the last base in the growing chain.

This means that the RNA grows in the five prime to three prime direction.

Transcription initiation does not go smoothly.

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.

The whole process of synthesis then starts again.

This process is known as abortive initiation.

After multiple rounds of abortive initiation, RNA polymerase breaks free of the promoter

and continues to synthesize the RNA in a more persistent way.

This phase is called promoter clearance.

RNA polymerase now forms a stable complex with the DNA and continues to synthesize the RNA.

At the same time, TF2H phosphorylates the C-terminal domain of the large subunits of RNA polymerase II.

This domain is called CTD.

This phosphorylation of CTD is key to the processing of the RNA to produce the mature transcript, and this is what happens next.

RNA Processing

During the RNA processing step, a complex of RNA processing enzymes called P-TEFB

binds to the phosphorylated CTD.

As the five prime end of the growing RNA comes out of the exit channel of RNA polymerase,

P-TEFB covalently attaches a guanosine cap to the five prime end of the RNA.

P-TEFB then attaches additional phosphate groups to the CTD, which causes transcription elongation, which is paused, to resume.

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.

Elongation

RNA polymerase has now transitioned into the elongation phase.

During this phase, the growing RNA strand comes out of the RNA polymerase exit channel as it continues to elongate.

Transcription bubble moves along with the RNA polymerase.

DNA base pairs are separated ahead of the transcribing polymerase to expose more of the template strand.

More bases in the wake of the polymerase re-anneal.

In this way, RNA polymerase can continue until it has transcribed the complete RNA.

Polymerase Stalling

RNA polymerase doesn't always make it to the end of the coding sequence in one smooth journey.

Instead, RNA polymerase can sometimes stall before reaching the end of the transcribed region.

The polymerase can be restarted by the binding of elongation factors.

But sometimes the RNA polymerase starts to backtrack.

This means that the polymerase slides backwards along with the transcription bubble.

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.

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.

This exposes a new three prime hydroxyl in the RNA that is correctly positioned in the active site.

Transcription can now resume as before.

Termination of Transcription

Transcription continues until RNA polymerase II reaches a sequence in the DNA

called a polyadenylation signal.

After RNA polymerase has transcribed through this signal, sequences in the RNA transcript associate with the RNA processing proteins

that are bound to the polymerase CTD.

These proteins cleave the RNA transcript and add a sequence of adenine nucleotides to the three prime end of the transcript.

This run of adenines is called a poly A tail.

Once the poly A tail is added, RNA polymerase dissociates from the DNA and the mature RNA is released.

At this point, transcription is terminated and an RNA transcript has been successfully produced.