Translation Regulation of N Expression

The importance of N as a regulatory protein is reflected in the number of ways the expression of the N gene is controlled. As the first gene of the pL operon, the N gene is regulated at the transcription level by CI and Cro repression pL (131). At the post-translation level, the Lon protease degrades N protein causing a relatively short protein halflife (68). Studies with l have identified two other regulatory mechanisms that modulate N expression, at the level of translation, through sites encoded in sequences in the 223 base long N-leader (figure 9-3). The first of these sequences is the nutL site and the second is a stem-loop structure sensitive to the double-stranded RNA-dependent endonuclease, RNaselll (24). This site, the N ribonuclease III (three) site, is referred to as N(RTS) (35).

The following discussion of these regulatory mechanisms is based primarily on three papers — Wilson et al. (181), Kameyama et al. (90), and Wilson et al. (182) — and is illustrated in figures 9-3 and 9-4. It was surprising to discover that the NUT site, which we have seen is a key element in the action of N in modifying the transcription complex, is also involved in controlling N synthesis at the level of translation. Even more confounding was the discovery that the very Nus products required for N's transcription antitermination activity are also required for this translation repression. This regulatory process relies on a u-a g-c u-a g-c u-a u-a g-c u-a g-c u-a


5'...CGCUCUUAAAAAAA agcauucaaag acaggagaauccagauggaugcacaa..3'

65 SD N

Figure 9-3 The N-leader transcript beyond PL.z The sequence is shown starting at the BOXA sequence of NUTL; numbers indicate distance from RNA start of PL. The structure of the RNaseIII site is shown with the position of cleavage sites marked by arrows. The N ribosome-binding site is underlined.

complicated interplay between the N(RTS), the NUT site, and the N ribosomal binding site. The RNaseIII-sensitive stem-loop structure can be viewed as being a central player in translation control. Located just prior to the N gene this large stem-loop structure in the RNA has two roles: first, it acts as a direct translation inhibitor, preventing ribosomes from easily binding at the N initiation region; second, it holds the Ngene initiation codon in the correct position relative to NUT for autorepression when N and the Nus factors bind to the NUT site. The stem is a substrate for RNaseIII (107), and its cleavage by RNaseIII prevents both types of repressive effects on N synthesis. Hence, translation repression is best observed in an RNaseIII mutant. In the absence of cleavage of the N(RTS), the newly expressed N protein acts by binding at the NUT site to block access to the N ribosome-binding site. This activity of N, like the antitermination activity, requires the participation of the Nus factors. Translation repression is specific for expression of N since translation of downstream genes is not affected. Cleavage by RNaseIII separates this ribosome-binding site from the NUT site and thus eliminates N repression. An N leader deleted for sequences encoding the entire N(RTS) maintains N-mediated translation repression. This shows that the stem structure itself is not essential for translation repression by N. RNaseIII does not prevent translation repression when the N(RTS) is not present because the processing site has been removed. Hence, RNaseIII's role in translation repression is due to its action at the N leader site.

In summary, the N(RTS) and RNase III action at that site regulate N expression by influencing translation initiation. The structure per se partially interferes with ribosome binding and N-mediated repression provides an additional block by sterically inhibiting ribosome binding. RNaseIII and N expression both increase with increasing growth rate (182) consistent with the hypothesis that RNaseIII activity stimulates N gene translation through cleavage of the inhibitory hairpin in a growth-rate-dependent manner. The relationship between RNaseIII activity and N expression can thus tie lambda development to the physiology of the host cell.

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