Coda

Transcription antitermination can serve two different roles useful to the phage. First, it allows the phage to acquire new genes even if the genes have associated transcription terminators. If an acquired gene locates within an operon, the antitermination mechanism enables transcription to reach downstream genes even if the acquired gene has an associated downstream terminator. For example, in phages carrying genes encoding Shiga toxin, even though the stx genes have an associated promoter, pstx, transcription of genes downstream of the stx genes only occurs from the pRi promoter, which is upstream of pstx (119, 170). Second, the antitermination system can serve as a regulator of gene expression. The regulation can be temporal and/or physiological.

In considering transcription antitermination in the lamb-doid phages, it is curious that these phages employ three different strategies to achieve antitermination. The PUT system appears to rely solely on an RNA structure. The N system relies on RNA sequences and structure as well as both phage- and host-encoded proteins. The Q system relies on a DNA binding site within the promoter, synthesis of a short transcript from that promoter, and a phage and a host protein. What advantages do these different strategies provide to the phage?

Antitermination systems that rely on interactions of sequences in the RNA are always found in the early operons, while those that rely on interactions at sequences in the DNA are found in late operons. We suspect that this arrangement of antitermination sequences is not fortuitous, but reflects some selective advantage to the phage. Regulation of gene expression of early functions is most important in temperate phage development because it is during the expression of these genes that the decision between lysogeny and lysis is made. We have presented studies showing how the N-NUT system provides regulation of gene expression at the levels of transcription and translation. In a subset of phages (those with N-NUT systems like that of H-19B), formation of an RNA structure per se within the NUT region, called the "reducer," appears to contribute to the effectiveness of the NUT site at directing antitermination. When the sequences encoding the reducer structure are removed, the effectiveness of the NUT site in directing N-mediated antitermination is only modestly reduced. In l, formation of a stem-loop structure per se in the N leader regulates the effectiveness of N-mediated antitermination in another way, by controlling the level of N expression. When present it serves to inhibit expression of N and its removal by RNase III allows higher levels of N to be expressed. Thus, modulation of the action of RNase III provides a mechanism to allow input of the physiological state of the bacterium into the regulation of N expression. The requirement for the action of a number of host proteins also provides additional potential targets for regulatory action.

The situation is different for late gene expression. Once the decision for lysis has been made, monitoring of late gene expression is unnecessary and expression can essentially be constitutive. The level of Q, which is determined by activities regulated by actions in the early operons, determines expression of late genes. Thus, it is likely that proper phage development requires less regulation of late gene expression. Although the l Q activity is enhanced by NusA in vitro, the activity of the Q of phage 82 is only modestly enhanced by NusA (137, 186). Moreover, none of the other Nus factors appear to contribute to Q action (7). Although NusA may allow some regulation of Q action at qut, there is obviously not the panoply of possible regulatory inputs that are available through the NUT RNA site.

Results of studies with phage HK022 appear to contradict the idea that the selective advantage of an RNA antiterminator is that it provides more opportunity for more regulatory inputs. The PUT RNA antiterminator located in the early operons of HK022 appears to function independently of either phage or host auxiliary proteins, unlike the N-Nus modulated NUT RNAs. If the PUT stem-loop and RNA Pol interact in the absence of other inputs and they alone are sufficient to direct processive transcription through downstream terminators, it would be difficult to argue that the antitermination mechanism in the early operon provides an important regulatory capability. PUT RNA is a component of every initiated transcript and therefore it would appear axiomatic that every RNA Pol becomes antitermination-proficient as soon as the put sequence is transcribed. In vivo genetic evidence suggests that only the RNA structure is essential for PUT-mediated antitermination. However, two observations showing that PUT-mediated antitermination can be more effective in vivo than in vitro suggest that, as yet, unidentified regulatory controls are exerted on the structure and/or function of PUT in vivo. Firstly, the antitermination activity observed with a DNA template having a put site is not as efficient in a pure transcription system with RNA Pol as it is with such a template in vivo. Secondly, some rpoC mutations cause a greater decrease in PUT effectiveness in vitro than in vivo (92,152). Experiments with l provide direct evidence that N plays a regulatory role in phage development (181). l forms turbid plaques on E. coli, and this indicates that some of the progeny phages formed in the localized infection go down the lysogenic route. However, the same l forms clear plaques if the E. coli lawn constitutively expresses N. This indicates that very few of the progeny phages go down the lysogenic route if N is present from the beginning of the infection. Hence, the timing and perhaps the level of expression of N is an important factor in determining how the phage develops.

We have already discussed how expression of N can be regulated; establishing how PUT is regulated or whether it is regulated awaits further work.

While many of the details of the action of l antiterminators have been elucidated, precisely how they enable RNA Pol to transcend terminators awaits further work. We hope that this chapter has provided new ways to think about this process that may serve to stimulate further experimentation.

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