NIVEL DE CONOCIMIENTO DE LAS MADRES

The mechanism of transcription is best understood in prokaryotes. The

extensive use of genetics and the relative ease with which proteins can be purified and

studied in vitro has generated a clear picture of the elongation process in E.coli.

Bacterial RNA polymerase has three functional domains - the front-end domain, the catalytic site, which binds RNA loosely (sitel), and the RNA-product binding site, which binds RNA tightly (site2) (Figure 1.4). Site 1 is filled as the RNA chain grows, and 5 to 10 bases can accumulate at site 1 without any forward motion of the polymerase. When site 1 is full, the polymerase becomes "strained" (see below). It reverts to the "relaxed" form when RNA from site one is suddenly released, taken up by

Figure 1.4. The 'inchworm' model of transcriptional elongation.

Polymerase movement is shown from left and right. Two RNA-binding sites in the polymerase molecule (shaded boxes) are coupled to DNA-binding sites in the original model (Chamberlin 1994); however, the DNA has been omitted for clarity. The catalytic site (black circle) is linked to the back end of the enzyme and the wavy line represents RNA. The two binding sites can move forward synchronously (monotonie movement) or asynchronously (inchworm movement). In the case of inchworm movement, the polymerase goes through a cycle of nucleotide addition without forward movement (A-C). This is followed by a jump forward (D). (A, D) represent the 'relaxed' conformation and (C) represents the 'constrained' conformation. The catalytic site can disengage in the strained conformation leading to arrest (E). (Adapted from Johnson and Chamberlin, 1994).

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site 2, and the polymerase jumps forward. Therefore the process of elongation is not a steady movement through the gene but has periods of steady monotonie progress (one base at a time) followed by jumps-like an inchworm, hence the name for this model (Chamberlin, 1994).

The inchworm model has been proved by footprinting the front end of the polymerase on DNA and comparing this to the catalytic site position as polymerase is moved along DNA one base at a time (Nudler et al., 1994). The results show that for much of the time, there is a constant distance of 18bp from the front of the polymerase to the catalytic site. However, some of the time this distance is reduced to 12bp indicating that the front end is held up while the catalytic site continues to add bases to the growing chain. In support of this, when polymerase is in the "strained" conformation with the front end stopped, nascent RNA is more susceptible to cleavage by addition of moderate doses of the transcription cleavage factor GreB, than when polymerase is in the relaxed conformation. In addition, cleavage releases larger fragments supporting the idea of a different conformation of polymerase with RNA more accessible.

This inchworming process is also dependent on the transcribed sequence. Furthermore, when polymerase transcribes a run of T residues that act as a terminator element, polymerase adopts a strained conformation before terminating transcription, suggesting that pausing of polymerase is a pre-requisite for termination (Nudler et al.,

1995).

Similar observations have been made in yeast. Yeast Pol II can bind RNA in a binary complex. These Pol II-RNA complexes were able to add nucleotides, and RNA could be cleaved by TFIIS (the mammalian equivalent of GreB), suggesting that, like bacterial RNA polymerase, RNA was bound at two positions separated by 10 nucleotides (Johnson and Chamberlin, 1994). The inchworm model may thus be representative of the eukaryotic polymerase.

RNA polymerase pauses and then may release the RNA transcript if transcription terminates. Some sites behave as particularly strong terminators in bacteria: for example the terminators just downstream of the start site of transcription

of the bacteriophage X pR and pL promoters. During the lytic phage X infection of

E.coli, the phage X N protein prevents termination at multiple sites in the transcribed regions from both the pR and pL promoters, allowing production of full-length RNA.

This allows E.coli RNA polymerase to transcribe the O and P genes (involved in the

replication of phage DNA) and the Q gene more efficiently. The Q gene encodes a

protein that prevents termination during transcription from the pR' promoter in the later stages of lytic infection (Greenblatt et ah, 1993).

N and Q function in different ways. The N protein is an arginine-rich RNA binding protein that is targeted to the transcribed region by sequence-specific binding to a region of RNA called the Nut (N utilisation) site. There are multiple contacts between N, a group of cellular proteins (NusA, NusB, NusG, and SIO, which all have roles in the modulation of termination and anti-termination), and RNA polymerase. N

is able to suppress pausing by RNA polymerase in vitro, but the mechanism is not

clear; one possibility is that N speeds up elongation by stabilising a conformation of the polymerase that does not permit pausing.

In contrast to N which acts via a Nut site on RNA, the Qut-Q utilisation- site is on DNA. The Qut site is needed for anti termination by Q lies between -26 and 4-18 of the late promoter pR'. Q protein recognises paused polymerase complex as a substrate and accelerates the polymerase out of the pause site, through the termination site (tR") and into the late transcribed genes. It is unclear how Q is able to stimulate elongation of the polymerase well beyond a region of DNA that it will bind. One possibility is that polymerase binds Q and continues its elongation with Q bound; alternatively Q is somehow able to alter the RNA polymerase conformation to render it functionally competent for elongation.

In summary, bacterial RNA polymerase elongation is not a steady movement through an operon, but moves and jumps depending on the sequence upstream of the polymerase. Either an RNA-binding or DNA-binding viral protein can increase the ability of polymerase to elongate through the transcribed region. The parallels with the eukaryotic systems are discussed below.