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PubMed1983

The tL2 cluster of transcription termination sites between genes bet and ral of coliphage lambda.

The major leftward transcription of bacteriophage lambda is controlled by several terminators (t), including tL1, tL2, tL3, and others. The tL2 termination site, which was placed by Salstrom and Szybalski (Virology 88, 252-260, 1978) between lambda genes bet and ral, was found to consist of a cluster of four leftward terminators. As not to change the numbering of other leftward terminators, these were designated as tL2a, tL2b, tL2c, and tL2d. As determined by S1 nuclease mapping of the tL2-terminated in vitro transcripts, the normally pL-initiated major leftward lambda transcription should encounter termination points at 1653 bp (tL2a; between genes Ea10 and ral), 2089 bp (tL2b; between genes cIII and Ea10), 2441-2442 bp, and 2483 bp (tL2c and tL2d; both within gene gam) from the sL startpoint (= +1). All terminators were cloned in a pBR322-derived plasmid between the p'R promoter and the galK gene, and their in vivo termination efficiencies are 69% (tL2a), 53% (tL2b), and 38% (tL2c + tL2d), measured as reduction of galK expression in rho+galK- hosts at 30 degrees. The tL2a and tL2b), terminators depend little on the rho factor, whereas the efficiency of tL2c + tL2d decreases from 38 to only 14% in the rho- host. When shifted to 42 degrees, the termination efficiency of tL2b decreases from 53 to only 36%, while the other tL2 terminators are much less affected by increasing the temperature. The calculated joint efficiency of the entire tL2 cluster is 90%, which is in perfect agreement with the 90% termination efficiency reported by Salstrom and Szybalski (1978) for tL2. However, the natural location of tL2c and tL2d within the actively translated gam gene may interfere with their termination function. Under in vitro conditions, tL2c and tL2d are active only in the presence of rho factor, whereas tL2a and tL2b do not require rho. The structure of the tL2 terminators resembles that of some other known termination sites: a perfect (8 bp for tL2a and tL2b) or imperfect (8-9 bp) dyad symmetry and a T6 (tL2a), T5 (tL2b), T4 (tL2c) or TTATT sequence (tL2d) toward the 3' end of the mRNA-like DNA strand.

Bacteriophage lambda↗
PubMed1979

Inceptor and origin of DNA replication in lambdoid coliphages. I. The lambda DNA minimal replication system.

In a pBR313-lambda dv hybrid plasmid system, stepwise deletion and serial cloning procedures have led to a functional dissection of the DNA replication region of lambdoid bacteriophages lambda, 434 and 21. A simple system for initiation of DNA replication has been detected within lambdoid replicator DNAs, which is active in the absence of several normal replication elements, including the origin of replication (ori) and product of gene O. This "minimal" (or "mini") initiation system depends on the p0 or substitute leftward promoter in conjunction with the newly discovered "inceptor" (ice) element, which is located within the cII gene. Even the fragments containing ori are unable to initiate replication in these hybrid plasmids as long as fragments containing ice are missing. The base sequence of ice resembles transcriptional terminators and it appears to control both termination of primer RNA and inception of daughter strand DNA synthesis. Initiation in the p0-ice mini system of lambda or 21 phages requires the gene P product. Hwever, mini replication of 434 DNA hybrid plasmids required neither O nor P proteins, although there are only two single-base changes in the 434 inceptor sequence. The mini system is repressed by the elements of the maximal lambda replication system, as described in the accompanying publication.

Bacteriophage lambda↗
PubMed1979

Inceptor and origin of DNA replication in lambdoid coliphages. II. The lambda DNA maximal replication system.

In pBR313-lambda dv hydrid plasmids a second system for initiation of DNA replication has been detected in lambdoid replicator DNAs (in the absence of the p0 promoter). The "maximal" (or "maxi") initiation system depends on the origin of replication (ori) sequence, in conjuction with the "inceptor" (ice) element located in the lambdoid cII genes. Only leftward, but not bidirectional, primer RNA synthesis seems to be initiated at ori in its newly defined boundaries, and it appears to be catalysed by dnaG-coded primase. Only if transcriptionally activated, will ori effectively initiate lambda specific, O and P-dependent "maximal" hybrid-plasmid replication. In addition, it will repress a complete lambda "minimal" initiation system in cis, i.e., if present on the same plasmid molecule. This newly discovered repressive activity of the ori system depends on only three factors: an intact left section of ori, the O product, and transcriptional activation of ori (rightward or leftward). A repressed minimal initiation system will regain its activity as soon as a segment carrying either part of the O gene or a promoter for transcriptional activation is delected from such a plasmid which was combining both the "mini" and "maxi" systems of lambda replication.

Bacteriophage lambda↗
PubMed1980

DNA sequence analysis of prm-mutations of coliphage lambda.

Nucleotide sequence changes associated with mutation of the prm promoter of bacteriophage lambda have been determined. Prm-mutations have been assigned to two classes. Class I mutations appear to affect the interaction of RNA polymerase with prm; six class I mutations affect four sites, located 14, 33, 38, and 39 bp preceding the prm transcription startpoint. Class II mutations appear to owe their Prm-phenotype to a change in OR, which could prevent activation of prm by repressor. All three class II mutations are in OR 1.

Bacteriophage lambda↗
PubMed1980

A biochemical assay for the transcription-antitermination function of the coliphage lambda N gene product.

An in vitro assay has been developed for the antiterminating activity of the N gene product (pN) of bacteriophage lambda, based on the N-dependent stimulation of trp mRNA synthesis from the DNA templates of lambda trp transducing phages. Using this assay system, we show (a) that the stimulation of trp mRNA synthesis by pN requires the cis-dominant nutL site on the lambda chromosome and (b) that pN can participate in vitro in the formation of functional antiterminating transcription complexes.

Bacteriophage lambda↗