rexB of bacteriophage lambda is an anti-cell death gene.

In Escherichia coli, programmed cell death is mediated through "addiction modules" consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of lambdarexB, one of the few genes expressed in the lysogenic state of bacteriophage lambda, prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3',5'-bispyrophosphate (ppGpp) and thus by amino acid starvation. lambdaRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of lambdaRexB through its action on the ClpP proteolytic subunit. We also propose that the lambdarex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the "survival operon" of phage lambda.

Regulatory implications of translational frameshifting in cellular gene expression.

The genetic code, once thought to be rigid, has been found to be quite flexible, permitting several different reading alternatives. One of these is translational frameshifting, a process programmed in the mRNA sequence and which enables a +1 or -1 shift from the reading frame of the initiation codon. So far, the involvement of translational frameshifting in gene expression has been described mainly in viruses (particularly retroviruses), retrotransposons, and bacterial insertion elements. In this MicroReview, we present a survey of the cellular genes, mostly in Escherichia coli, which have been found to be expressed through a translational frameshifting process, as well as a discussion of the regulatory implications of this process.

Energy-dependent degradation of lambda O protein in Escherichia coli.

Protein O of bacteriophage lambda is a short-lived protein which has a key role in the replication of the phage DNA in Escherichia coli. Here we present evidence that lambda O degradation is energy dependent: it is impaired by cyanide and alpha-methylglucoside, both of which inhibit cellular energy metabolism. Removal of these inhibitors restored the degradation of lambda O. Our experiments suggest that limited amounts of cellular energy are sufficient to support lambda O degradation. In addition, degradation of lambda O protein is prevented by a mutation in the E. coli clpP gene, but not by a mutation in the clpA gene. These results suggest that the ClpP protease is involved in the energy-dependent degradation of the lambda O protein.

Translational introns: an additional regulatory element in gene expression.

The linear expression of a gene can be interrupted by the well-known RNA introns and the recently discovered protein introns. In both cases, splicing mechanisms physically excise the unexpressed segments. In this article we describe a third category of introns that we call 'translational introns'. These functional introns are not excised through a splicing mechanism; instead, the translational machinery bypasses a segment of the coding sequence of an mRNA. We suggest that 'translational introns' are part of a regulatory mechanism that may sense changes in the rate of translation and thereby control the ratio of alternative gene products.

An additional function for bacteriophage lambda rex: the rexB product prevents degradation of the lambda O protein.

The rex operon of bacteriophage lambda excludes the development of several unrelated bacteriophages. Here we present an additional lambda rexB function: it prevents degradation of the short-lived protein lambda O known to be involved in lambda DNA replication. We have shown that it is the product of rexB that is responsible for the stabilization of lambda O: when a nonsense mutation is present in rexB, lambda O protein is labile; suppression of the mutation by the corresponding nonsense suppressor causes partial restabilization of lambda O. lambda rexB also stabilizes lambda O in trans. We discuss our results in relation to the function of rexB in lambda DNA replication and its role in the protein degradation pathways of bacteriophage lambda.

Stop is not the end: physiological implications of translational readthrough.

The translational readthrough mechanism permits the occasional misreading of termination codons by normal charged tRNAs causing extended translation beyond the stop signal. In both prokaryotes and eukaryotes translational readthrough is involved in the regulation of gene expression, as for example in the synthesis of the enzyme reverse transcriptase of the Murine Leukemia Virus (MuLV) (Yoshinaka et al., 1985). Here we particularly deal with the sensitivity of the translational readthrough process to two parameters which are affected by changes in physiological conditions: (1) fluctuations in the concentration of readthrough tRNAs and (b) The affinity of the tRNAs to termination codons. We also discuss the possible role of translational readthrough during major changes in cell physiology.

Escherichia coli tryptophan operon directs the in vivo synthesis of a leader peptide.

Here we report the identification of the Escherichia coli trp leader peptide synthesized in vivo. We identified the peptide in UV-irradiated maxicells by selective labeling with radioactive amino acids which are included in the predicted sequence of this peptide. Our results support the hypothesis that translation of the peptide-coding region of the leader RNA has a role in the mechanism of attenuation of biosynthetic operons in general and in the E. coli trp operon in particular.

Modulation of Escherichia coli tryptophan (trp) attenuation by the UGA readthrough process.

We constructed plasmid pAtrp46 in which lacZ gene expression is regulated by the attenuator of the Escherichia coli tryptophan (trp) operon. The attenuation of trp, which occurs in the presence of an excess of tryptophan, is reflected by a decrease in the expression of the lacZ gene of pAtrp46 in a trpR- strain. Experiments with pAtrp46 further support our previous results (Engelberg-Kulka et al. 1982b) that suppression of a UGA termination codon by normal charged tRNATrp, a process called UGA readthrough, is a necessary mechanism in trp attenuation. Our experiments also suggest that plasmid pAtrp46 is useful for studies of other aspects of trp attenuation.

Effect of an Escherichia coli traD (ts) mutation on MS2 RNA replication.

We have continued our studies on the effect of high temperature (42 degrees C) on the development of RNA bacteriophage MS2 in the temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39 carrying a traD (ts) mutation. At 42 degrees C, mutant cells permit the penetration and translation of phage MS2 RNA but do not permit MS2 RNA replication. We suggest a role for the traD (ts) mutation in MS2 RNA replication.

Genetic analysis of a streptomycin-resistant Escherichia coli mutant temperature-sensitive for nonsense suppression.

Continuing the genetic and biochemical characterization of the streptomycin-resistant Escherichia coli mutant LD1, we confirmed that LD1 is temperature-sensitive for suppression of nonsense codons, and that this phenotype of the mutant and its streptomycin-resistance are genetically linked and are probably caused by a single mutation, strA(LD1). We also isolated a spontaneous revertant, called LD1-R, which partially relieves the restriction of nonsense suppression caused by the strA(LD1) mutation. LD1-R is derived by an additional mutation (revA) which is closely linked to strA(LD1). We further demonstrate that the weak suppression of a lacZUGA mutation in a suppressor-free strain, which probably takes place by normal tRNA1rp, can be detected by the use of the chromagenic substance x-gal (5-Bromo-4-chloro-3-indolyl-beta-D-Galactopyranoside).

Studies on the involvement of the UGA readthrough process in the mechanism of attenuation of the tryptophan operon of Escherichia coli.

We asked if UGA suppression by charged tRNATrp, a process called UGA readthrough, is involved in the mechanism of attenuation of the tryptophan (trp) operon in Escherichia coli. For this purpose we used two mutations: strA(LD1) which causes restriction of UGA readthrough, and revA which partially overcomes the restriction of UGA readthrough caused by strA(LD1)(Engelberg-Kulka et al. 1982). trp attenuation was monitored by the regulation of the synthesis of the trp operon enzyme anthranilate synthetase (ASase) in trpR strains. We showed that the strA(LD1) mutation causes a significant increase in the level of synthesis of ASase in the presence of an excess of tryptophan, while the revA mutation reverses this effect, indicating that transcription termination at the trp attenuator site is relieved by restriction of UGA readthrough. Based on our results and the sequence data of the trp leader RNA of E. coli (Oxender et al. 1979), we offer a model for the involvement of the UGA readthrough process in trp attenuation. We suggest that the UGA readthrough process permits trp attenuation to respond to slight changes in the cellular concentration of charged tRNATrp.

Escherichia coli traD(Ts) mutant temperature sensitive for assembly of RNA bacteriophage MS2.

We report here a study on the temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39, carrying a traD(Ts) mutation, which is also temperature sensitive for group I RNA phages (MS2, f2, and R17). It is shown that, when the mutant was infected with MS2 at 42 degrees C, phage RNA replicated; a 27S MS2 RNA and phage proteins were synthesized. However, neither PFU nor physical MS2 particles were formed, showing that phage assembly was inhibited. In addition, the high temperature affected the membranes of the host mutant: the mutant was hypersensitive to chemicals, and the electrophoretic pattern of the membranal proteins was modified. We suggest that the pleiotropic effects of the traD mutation on MS2 assembly and DNA transfer during conjugation were a result of the changes in the membrane of the mutant.