The carboxy-terminal 14 amino acids of phage lambda N protein are dispensable for transcription antitermination

Abstract

The analogous N proteins encoded by lambdoid bacteriophages X, 21, and 22 are very different in amino acid sequence, except at their carboxy-terminal ends. Since NX remains functional despite the deletion of most of its terminal region of homology to N21, that region of homology cannot represent a region of conserved function.The NA protein, encoded by the bacteriophage A N gene, is required to prevent transcription of X from being prematurely terminated (reviewed in reference 13). Subsequent to transcription initiation in early operons of X, the NA protein responds to the nut (= boxA + boxB) sequences (22) within the early transcripts and causes a transcription complex to be assembled. This complex is constituted of NA protein in conjunction with RNA polymerase and four Nus proteins from the Escherichia coli host (1,15,17,23). The transcription complex is unresponsive to signals in the genome that would otherwise cause transcription to be terminated.As part of my effort to characterize the NA protein and understand the means of its interactions, I have generated deletions into the 3' end of its coding gene, NA. Sequencing of this gene (11) had shown that NA is a protein of only 107 amino acids. Sequencing of analogous genes from related phages 21 and P22 had revealed somewhat smaller proteins barely if at all related in amino acid sequence, except that NA and N21 are identical in 21 of their 32 carboxy-terminal amino acids ( Fig. 1) (8). Other possible commonalities among N proteins, including an amino-terminal arginine-rich cluster, have been noted and are under study (8,10,16).Since NA, N21, and N22 are each required by their respective phages to overcome the transcription termination signals within each genome, it is of interest to understand the nature of proteins that differ so dramatically in amino acid sequence yet accomplish the same function. In particular, it is important to learn whether the section of common sequence at the carboxy termini of NA and N21 is significant to antitermination function.Two plasmids used to supply and detect N. For the purpose of generating deletions into the NA gene while monitoring for NA function, it was convenient to use the two-plasmid system developed to visualize antitermination in vivo (9). The N-related segments of the two plasmids were switched with respect to the carrier plasmids so that N would be present in a higher copy number on the pBR322-derived member (Fig. 2). The N supplier plasmid has the isopropyl-,-D-thiogalactopyranoside (IPTG)-inducible ptac promoter upstream from N*A, where N*A differs from wild-type NA by four silent mutations introduced into the amino-terminal half of N+A to provide additional unique restriction targets (10). The ptac-N* segment was introduced into a pBR322 derivative from which the Tetr gene had been deleted (leaving the Ampr gene) to yield pBR-ptacN*A. The N tester plasmid for visualizing N-mediated antitermination contains a synthetic operon, TAT13: ptac precedes a synthetic but functional boxA-boxB recognition sequence for N...

A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination

“…Even though the mutagenesis process was random, we obtained point mutations only in the RNA-binding domain of N. Therefore, we hypothesized that the C-terminal RNAP-binding domain of N (CTD) (Supplementary Figure S1A) may not be functionally important for anti-termination on this construct. Dispensability of the c-terminal 14 amino acids of the λN protein was observed earlier (32). To test this, we made several deletions in the CTD of H-19B N. Compared with the point mutants described earlier, the deletions in the last 27 amino acids of H-19B N did not show severe defect (≤1.5-fold compared with the WT) in anti-termination.…”

Conservation and diversity in the immunity regions of wild phages with the immunity specificity of phage λ

“…Differences in this part of N suggest the possibility that interactions with RNA polymerase may differ between this N and that of λ. It is also known that the C‐terminal 14 residues of the λ N protein are dispensable for function, at least when the protein is present in vivo in high amounts (Franklin, 1992), implying some flexibility in rearrangements in this interval. [A recent genome sequence (Chen et al ., 2006) from E. coli UTI89 included a prophage bearing a putative N gene with all but the first 33 residues of 21 N , suggesting the possibility of still further flexibility.]…”