The below mentioned article provides a study note on the transcription in prokaryotes.
In prokaryotic organisms, transcription occurs in three phases known as initiation, elongation and termination with the help of single RNA polymerase.
RNA is synthesized by a single RNA polymerase enzyme which contains multiple polypeptide subunits.
In E. coli, the RNA polymerase has five subunits:
two a, one p, one P’ and one a subunit (α2ββ’σ). This form is called the holoenzyme. The σ subunit may dissociate from the other subunits to leave a form known as the core enzyme.
These two forms of the RNA polymerase have different roles in transcription. The σ subunit is required for the interaction with the sigma factor. The sigma factor recognizes the start signal of DNA and directs the binding of the enzyme to the initiation site on DNA template. The binding of RNA polymerase to DNA involves the β subunit.
Initiation involves binding of RNA polymerase to the promoter site.
Promoter Site for Initiation:
Transcription cannot start randomly but must begin specially at the start of a gene. Signals for the initiation of transcription occur in the promoter sequence which lies directly upstream of the transcribed sequence of the gene.
The promoter contains specific DNA sequences that act as points of attachment for the RNA polymerase. The exact sequences can vary between promoters but ail conform to an overall pattern known as the consensus sequence.
In E. Coli, two sequence elements, -10 sequence and -35 sequence, are recognized by the RNA polymerase. The consensus -10 sequence, also called the “Pribnow box” is TATAAT and the consensus -35 sequence, also called the “recognition sequence” is TTGACA. (Fig. 16.2).
The σ subunit of the RNA polymerase is responsible for recognizing and binding the promoter, probably at the -35 box. In the absence of the σ subunit, the enzyme can still bind to DNA but binding is more random.
Initiation of RNA Synthesis:
When the enzyme binds to the promoter, it initially forms a closed promoter complex in which the promoter DNA remains as a double helix. The enzyme covers about 60 base pairs of the promoter including the -10 and -35 boxes.
To allow transcription to begin, the double helix partially dissociates at the -10 box, which is rich in weak A-T bonds, to give an open promoter complex. The σ subunit then dissociates from the open promoter complex leaving the core enzyme. At the same time the first two ribonucleotides bind to the DNA, the first phosphodiester bond is formed and transcription is initiated (Fig. 16.3).
During elongation, the RNA polymerase moves along the DNA molecule, melting and unwinding the double helix as it progresses. The enzyme adds ribonucleotides to the 3′ end of the growing RNA molecule with the order of addition determined by the order of the bases on the template strand.
In most cases, a leader sequence of variable length is transcribed before the coding sequence of the gene Is reached. Similarly, at the end of the coding sequence, a noncoding trailer sequence is transcribed before transcription ends. During transcription, only a small portion of the double helix is unwound at any one time.
The unwound area contains the newly synthesized RNA base-paired with the template DNA strand and extends over 12-17 base. The unwound area needs to remain small because unwinding in one region necessitates over winding in adjacent regions and this imposes strain on the DNA molecule.
To overcome this problem, the RNA is released from the template DNA as it is synthesized allowing the DNA double helix to reform (fig. 16.4).
Chain elongation takes place by addition of activated ribonucleoside triphosphates (ATP, UTP, GTP and CTP) to one strand of the DNA template. For each nucleotide added to the growing RNA chain, pyrophosphate (PPi) is given off. This is rapidly hydrolysed to inorganic phosphate (Pi).
The synthesis of the RNA chain is energized by expenditure of energy in the form of pyrophosphates. For each nucleotide monomer added to the chain, two high energy phosphates are expended.
The entire reaction may be summarized as follows:
Elongation of the RNA chain takes place by means of the core enzyme which moves along the DNA template. During transcription, RNA is synthesized by the polymerization of ribonucleotide triphosphate subunits (ATP, UTP, GTP, CTP).
The 3′-OH of one ribonucleotide reacts with the 5′ phosphate of another to form a phosphodiester bond. The transcript is synthesized in the 5′ →3′ direction but because the chain must be antiparallel for base pairing, the template strand runs in the opposite, 3’→5′ direction.
RNA Chains Grow in the 5’→3′ Direction:
If RNA chains are synthesized in the 5’→3′ direction, then the first nucleotide should have triphosphate group (P~P~P). If, on the other hand, the chain grows in the 3’→5′ direction, then the triphosphate group would be on the nucleotide at the growing end. It has been found that the triphosphate group is attached to the first nucleotide at its 5′ end and a free hydroxyl group at the 3′ end.
This shows that growth takes place in the 5′ →3′ direction.
Only One DNA Strand of a Gene Transcribes mRNA:
In double stranded DNA, a given gene is transcribed from one of the two strands. The transcribed RNA is complementary to only one of the two strands. All the transcribed genes need not, however, be on one strand of the DNA double helix.
One gene may transcribe mRNA from one strand while another transcribes from the other strand. The two strands or the double helix are called the template and the non-template strands. RNA is produced using the template strand and the RNA molecule synthesized is a copy of the non-template strand (Fig. 16.5), also named the sense (+) strand or the coding strand. The RNA molecule synthesized is called a transcript.
The termination of transcription occurs non-randomly and takes at specific points after the end of the coding sequence. In E. coli, termination occurs at sequences known as palindromes. These are symmetrical about their middle such that the first half of the sequence is followed by its exact complement in the second half.
In single-stranded RNA molecules, this feature allows the first half of the sequence to base pair with second half to form what is known as a stem-loop structure (Fig. 16.6). These appear to act as signals for termination. In some cases, the stem-loop sequence is followed by a run of 5-10. As in the DNA which form weak A-U base pairs with the newly synthesized RNA.
It is thought that the RNA polymerase pauses just after the stem-loop and that the weak A-U base pairs break causing the transcript to detach from the template.
In other cases, the run of As is absent and a different mechanism occurs based on binding of a protein called Rho (p) which disrupts base-pairing between the template and the transcript when the polymerase pauses after the stem-loop. The termination of transcription involves the release of the transcript and the core enzyme which may then re-associate with the o subunit and go on to another round of transcription.