Script for RNA Polymerase II Transcription Movie

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 transcription.

The start of the coding sequence is known as the transcription start site. To the 5’ 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.

The first step in the assembly of the transcription initiation complex is the binding of the transcription factor, TFIID, to the TATA box, which is one of the core DNA promoter elements. TFIID 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 TFIIB is followed by the binding of TFIIA and TFIIB. TFIIB binds to the core promoter element called the BRE, as well as contacting TFIID.

In the next step, the RNA polymerase II core enzyme is recruited, together with TFIIF. Finally, TFIIE and TFIIH 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 upon TFIIH, 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 ribonucleoside triphosphates enter through the funnel region of RNA polymerase and form base pairing interactions with the template strand. RNA polymerase catalyzes the addition of nucleotides to the 3’ hydroxyl of the last base in the growing chain; this means that the RNA grows in the 5’ to 3’ 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, TFIIH phosphorylates the C-terminal domain of the large subunits of RNA polymerase II. This domain is called CTD. This phosphorylation step is key to what happens next, in which processing of the RNA to produce the mature transcript is coupled to transcription.

A complex of RNA-processing enzymes called p-TEFb now binds to the phosphorylated CTD. As the 5’ end of the growing RNA comes out of the exit channel of RNA polymerase, p-TEFb covalently attaches a guanosine cap to the 5’ end of the RNA. P-TEFb then attaches additional phosphate groups to the CTD, which causes transcription elongation to resume. The more highly phosphorylated CTD also can recruit additional RNA processing enzymes that will be needed to complete processing of the transcript at the end of the transcription cycle.

RNA polymerase has now transitioned into the elongation phase, and the growing RNA strand comes out of the RNA polymerase exit channel as it continues to elongate. The transcription bubble moves along with RNA polymerase: DNA base pairs are separated ahead of the transcribing polymerase to expose more of the template strand, while bases in the wake of the polymerase re-anneal. In this way, RNA polymerase can continue until it has transcribed the complete RNA.

In some cases, though, RNA polymerase stalls before reaching the end of the transcribed region. The polymerase can be re-started 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 3’ end of the growing transcript to become unpaired and to come out through the funnel region of RNA polymerase.

The transcription factor, TFIIS, can rescue the stalled polymerase by binding in the funnel region and promoting cleavage of the 3’ end of the RNA that is unpaired. This exposes a new 3’ hydroxyl in the RNA that is correctly position in the active site. Transcription can now resume as before.

Transcription continues until RNA polymerase II reaches a sequence in the DNA called a terminator. After RNA polymerase has transcribed through the terminator, 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 3’ end of the transcript. This run of As is called a polyA tail. RNA polymerase dissociates from the DNA and the mature RNA is released.

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