Are Dispensable for Transcription Antitermination

The NA protein, encoded by the bacteriophage AN 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 NA (6), upstream reporter genephoA, a tandem array of strong terminators, and downstream reporter gene lacZ with a crippled translation start site. This TAT13 N tester segment was ligated into pACYC184, replacing the Tetr gene but leaving the Camr gene, to yield pAC-TAT13. The E. coli host for these plasmids is strain N567 (12), with deletions of recA, phoA, and lacZ but including mutation lacIq (providing excess repressor to ptac) and the four wild-type nus genes whose protein products are essential for N function. When the two compatible plasmids are introduced into N567, the lacZ gene of the tester plasmid is expressed when, and only when, the N* gene of the second plasmid is functional (9). The expression of lacZ can be observed with high sensitivity in bacterial colonies by the release of the blue hydrolysate of 5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside (X-Gal) added to the medium. The product of lacZ, P-galactosidase, can also be assayed in permeabilized cells (20) and is found, at levels of <200 U per unit of optical density at 600 nm, to be proportional to the blueness of colonies growing in the presence of X-Gal. The reassortment of N tester and N supplier segments with respect to carrier plasmids pACYC184 and pBR322 did not significantly affect the performance of this system for the in vivo detection of N antitermination function (Table 1). Deletions into the C terminus of N*x. To provide suitable sites for the initiation of unidirectional deletions intoN (14), a synthetic polylinker, with ends complementary toXbaI-cut ends and with internal targets for SacI and StuI, was added at the unique XbaI target just downstream from N*A. Prior to this addition, the SacI site upstream fromNwas removed by cutting with SacI and filling in with T4 DNA polymerase before religation. The resulting plasmid, pBR-ptacN*AL, is shown in Fig. 2. Deletions into N* of pBR-ptacN*AL were made as directed by Hennikoff (14) by cutting at the unique StuI site to yield a blunt end susceptible to digestion by exonuclease III, and at the unique SacI site to yield a recessed 5' end protected from exonuclease III action. The ends of the excised DNA were made blunt and religated. A blunt-ended synthetic oligonucleotide, related by permutation to the oligonucleotide used to provide SacI and StuI, was added during ligation in a second experiment to provide translation stops in all reading frames and close to N*. The sequence of this polylinker follows, with translation stop codons TAA and TAG emphasized: strand 1, 5' GTAATGATCAGA TCTAAGGCCTAGAGCTCATATGCATTAC (boldface regions are reading frames 2, 3, and 1, respectively); strand 2, 5' GTAATGCATATGAGCTCTAGGCCTTAGATCTGATC ATTAC (boldface regions are reading frames 2, 3, and 1, respectively).