Coliphage λnutL−: A unique class of mutants defective in the site of gene N product utilization for antitermination of leftward transcription

Salstrom, John S.; Szybalski, Waclaw

doi:10.1016/0022-2836(78)90156-0

Cited by 201 publications

(94 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Because tat shares structural and functional features with bacteriophage k N antiterminator (Franklin and Bennett 1979), which also does not bind to its N-utilization (nut) site, but does bind to a transcription complex that pauses at the n u t site (Barik et al 1987), it is possible that tat will bind only to its target sequence in the presence of RNA polymerase II and associated proteins. In a further analogy with HIV, the n u t site forms an RNA stem-loop, and a single nucleotide substitution in the loop abolishes antitermination by N (Rosenberg et al 1978;Salstrom and Szybalski 1978;Das and Wolska 1984).…”

Section: Discussionmentioning

confidence: 99%

Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

Selby¹,

Bain²,

Luciw³

et al. 1989

Genes Dev.

359

349

View full text Add to dashboard Cite

The human immunodeficiency virus (HIV-1)-encoded trans-activator (tat) increases HIV gene expression and replication. Previously, we demonstrated that tat facilitates elongation of transcription through the HIV-1 long terminal repeat (LTR) and that short transcripts corresponding to prematurely terminated RNA are released and accumulate in the absence of tat. Here, using a transient expression assay, we tested clustered and compensatory mutations, as wall as 3' deletions, in the trans-acting responsive region (tar) and observed that the primary sequence in the loop and secondary structure in the stem of the stem-loop in tar are required for trans-activation by tat. Insertions in the 5' region of tar revealed that tar must be near the site of HIV-1 initiation of transcription for trans-activation by tat. Deletions (3') and an insertion in tar demonstrated that an intact stem-loop is required for the recovery of prematurely terminated transcripts. Short and full-length transcripts were observed also with HIV type 2 (HIV-2) in the absence and presence of tat, respectively. We conclude that an intact stem-loop in tar is essential for trans-activation by tat and that initiation of transcription by HIV-1 promoter factors and elongation of transcription by tat are coupled.

show abstract

Section: Discussionmentioning

confidence: 99%

Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

Selby¹,

Bain²,

Luciw³

et al. 1989

Genes Dev.

359

349

View full text Add to dashboard Cite

show abstract

“…Homologies in DNA sequence have been noted between portions of the phage X nut sites, which are the proposed recognition sites for the X N gene product and the NusA protein (26,27), and the leader regions of all seven E. coli rRNA operons (ref. 5; E. Morgan, personal communication).…”

Section: Discussionmentioning

confidence: 99%

Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nusB5 mutant of Escherichia coli.

Sharrock¹,

Gourse²,

Nomura³

1985

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The nusB5 mutant of Escherichia coli was originally selected for reduced ability to support the antitermination of transcription that is mediated by the gene N product of bacteriophage X. By analyzing pulse-labeled RNA with an RNA-DNA ifiter hybridization technique, we have shown that, in the nusB5 mutant, the ratio of promoterproximal rRNA transcripts to promoter-distal transcripts is increased at least by a factor of 1.6; that is, in the absence of the functional nusB gene product, premature transcription termination takes place within rRNA operons. These results demonstrate that rRNA transcription in E. coli utilizes an antitermination mechanism that has at least one factor in common with the phage X system, the nusB gene product. We have also observed that the transcription initiation frequency at rRNA promoters is increased in the nusB5 strain and that this strain accumulates 30S and 50S ribosomal subunits at approximately the same rate as the parent. Thus, it appears thatE. coli compensates for premature termination of rRNA transcription by derepressing rRNA operon expression. The increase in rRNA promoter activity in the nusB5 mutant is accompanied by a parallel derepression of synthesis of tRNAs that are not encoded by rRNA operons. These results are consistent with a model for negative feedback regulation of rRNA and tRNA synthesis by products of rRNA operons.In Escherichia coli, transcription often terminates prematurely within polypeptide-encoding operons that contain nonsense mutations ("polar" mutations), presumably because the absence of translation exposes termination signals that are not normally active (1). Each of the seven rRNA operons of E. coli encodes =5000 bases of untranslated RNA that is processed to form the 16S, 23S, and 5S rRNAs as well as spacer and distal tRNAs (2, 3). It has been proposed that an antitermination mechanism functions during rRNA transcription to prevent premature termination within these operons (4, 5). In fact, it has been shown experimentally that termination sites within transposons and insertion elements (4,(6)(7)(8) Since the antitermination mechanism proposed for E. coli rRNA transcription may be similar to the phage X system (see ref. 5), we studied rRNA transcription in various nus mutants. The results presented in this paper show that premature transcription termination in fact takes place within rRNA operons in the nusB5 mutant. In addition, we analyzed the way in which regulation of rRNA and tRNA expression is altered in response to nusB5-induced premature termination of rRNA transcription. The results of these experiments are consistent with the feedback regulation model for rRNA and tRNA synthesis proposed (18, 19). (22). This probe covers the region from the fourth base downstream of the rrnB P2 start site to the 83rd nucleotide of mature 16S RNA (see Fig. 1). All other methods are described in the figure and table legends. MATERIALS AND METHODS RESULTS Premature Transcription Termination Within rRNAOperons Caused by the nusB5 Mutation. To stu...

show abstract

“…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).…”

mentioning

confidence: 99%

“…Furthermore, A93A and A93A' show the same relative activity as N* when tested for function against pAC-TAT13 having point mutations in the loop of boxB (Table 1). boaxB is an interrupted palindrome whose loop sequence has been found critical to N function (6,22); single-nucleotide changes at all five positions of the boxB loop impair or negate N function, suggesting that the 5-nucleotide boxB loop may be the site of a specific interaction between N and boxB (6). Apparently N with a carboxy-terminal deletion is not altered in its interaction with the boxB loop.…”

mentioning

confidence: 99%

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

Franklin

1992

J Bacteriol

View full text Add to dashboard Cite

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...

show abstract

Coliphage λnutL−: A unique class of mutants defective in the site of gene N product utilization for antitermination of leftward transcription

Cited by 201 publications

References 55 publications

Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nusB5 mutant of Escherichia coli.

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

Contact Info

Product

Resources

About