Second Site Suppressors

Mutations in E. coli and l have been obtained that suppress the effect of nus mutations on N action. Certain second-site mutations in the E. coli nusB gene as well as mutations in the l N gene and nut site enhance the action of N in E. coli carrying either the nusA1 or nusE71 mutations (53, 150,171).

The NUT Site (see figure 9-1)

Analysis of a cis dominant mutation that blocks the action of N in the pL operon led Salstrom and Szybalski (148) to identify the nut site in the pL operon. Based on this observation, the nut site in the pR operon was identified (146). Identification of a frameshift mutation in cro that allows translation past the normal translation stop in the cro gene and thereby blocks N action in the pR operon provided the first evidence that N action, at least in part, works through the NUT RNA (126).

The NUT site has three elements: BOXA and BOXB, a stem-loop structure, and between them a spacer region (figure 9-2) (52). As will be discussed in detail below, the N and Nus proteins assemble on the NUT site of the nascent RNA and modify RNA Pol to a termination- resistant form. Transcription initiating at pR is modified by N as it passes through the nutR site, causing RNA Pol to transcend the tR1 and nin terminators. This allows transcription of the downstream O and P replication genes, the nin region, and the Q gene. Similarly transcription from pL is modified by N as it passes through the nutL site, causing RNA Pol to transcend downstream terminators.

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