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H Engelberg-Kulka

Publications and source records attributed to H Engelberg-Kulka.

At least 37 records · Page 2Linked to original sources
PubMed1993

Frameshifting in the expression of the E. coli trpR gene occurs by the bypassing of a segment of its coding sequence.

The E. coli trpR gene encodes the 108 amino acid long trp repressor. We have previously shown that a +1 frameshifting event occurs during the expression of trpR. Here we show that the transition from the 0 to the +1 frame of trpR occurs by the bypassing of a 55 nt long segment of the trpR+1-lacZ mRNA. This bypassing event is not pretranslational, and it probably takes place during translation. Two adjacent elements are required: a specific sequence of trpR, which must be preceded by a nonspecific 5' end longer than 10 translatable codons. Unique to trpR-lacZ bypassing is that the 55 nt long region must be translated in frame 0 to enable bypassing into the +1 frame. Translational bypassing as a newly discovered mechanism of gene expression is discussed, and the possible existence of translational introns is suggested.

Amino Acid Sequence↗
PubMed1993

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.

Base Sequence↗
PubMed1993

Frameshifting in the expression of the Escherichia coli trpR gene is modulated by translation initiation.

The Escherichia coli trpR gene encodes the 108-amino-acid-long Trp repressor. We have shown previously that a +1 frameshifting event occurs during the expression of trpR, resulting in the synthesis of an additional (+1 frame) polypeptide. Using trpR-lac'Z fusions, we have recently found that the transition from the 0 to the +1 frame occurs via the bypassing of a 55-nucleotide-long segment of the trpR+1-lac'Z mRNA (I. Benhar, and H. Engelberg-Kulka, Cell 72:121-130, 1993). Here we show that the frequency of trpR frameshifting (or bypassing) can be regulated both in vivo and in vitro. This frequency is inversely proportional to the rate of initiation of translation of the trpR gene. Hence, modulating the level of translation initiation affects the frequency of frameshifting.

Bacterial Proteins↗
PubMed1993

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.

ATP-Dependent Proteases↗
PubMed1992

Influence of codon context on UGA suppression and readthrough.

We studied the influence of the codon context on UGA suppression by a suppressor tRNA and on UGA readthrough by a normal tRNA in Escherichia coli. This was done by a series of constructs where only the immediate context of the TGA codon was varied by only one nucleotide at a time. For both UGA suppression and UGA readthrough the codon context had a similar influence according to the following rules. (1) The nature of the nucleotide immediately adjacent to the 3' side of the UGA is an important determinant; at that position the level of UGA translation is influenced by the nucleotides in the order A greater than G greater than C greater than U. (2) At extremely high or low levels of UGA translation this influence of the adjacent 3' nucleotide is not seen. (3) In all cases, the nature of both the nucleotide immediately adjacent to the 5' side of the codon and that following the base adjacent to the 3' side of the codon have little effect, if any, on UGA translation. The varying influence of the codon context effect on UGA translation is discussed in relation to its role in gene expression.

Base Sequence↗
PubMed1992

Frameshifting in the expression of the Escherichia coli trpR gene.

The trpR gene of Escherichia coli carries an open reading frame that encodes the trp repressor, 108 amino acids long. Here we show that translation of an additional (+1) reading frame of trpR occurs both in vivo and in vitro. This results in the synthesis of a stable +1 frame polypeptide. Using site-specific mutagenesis, immunological techniques and amino acid sequencing we have found that the N-terminus of the +1 frame product and that of the known 0 frame product are identical but that their C-termini differ. Our results are discussed in relation to the role of natural frameshifting as a regulatory mechanism of gene expression in general, and with respect to tryptophan biosynthesis in particular.

Amino Acid Sequence↗
PubMed1991

A procedure for amino acid sequencing in internal regions of proteins.

We describe a novel procedure for determining the amino acid (aa) sequence of the internal regions of proteins. This procedure has been implemented by directly determining the sequence of aa 65-75 of the product of the trpR gene of Escherichia coli, the trp repressor. This method is based on the insertion of the cleavage site of a specific protease (factor Xa) into the protein immediately before the region to be sequenced by Edman degradation. The simplicity of the procedure makes it appealing for studies of protein structure-function relationships, and of the expression of genetic information. The method is particularly useful when there is ambiguity concerning the co-linearity of the aa and nucleotide sequences.

Amino Acid Sequence↗
PubMed1991

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.

Bacteriophage lambda↗
PubMed1988

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.

Adaptation, Physiological↗
PubMed1986

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.

Amino Acid Sequence↗
PubMed1984

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.

Base Sequence↗
PubMed1983

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.

Coliphages↗
PubMed1982

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).

Codon↗
PubMed1982

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.

Anthranilate Synthase↗
PubMed1981

UGA suppression by normal tRNA Trp in Escherichia coli: codon context effects.

The nucleotide sequences at the 3' side of in-phase UGA termination codons in mRNAs of various prokaryotic genes were re-examined. An adenine (A) residue is found to be adjacent to the 3' side of UGA in mRNAs which code for readthrough proteins by the suppression of UGA by normal Escherichia coli tRNA Trp. It is suggested that the nature of the nucleotide following a UGA codon determines whether the UGA signals inefficiently or efficiently the termination of polypeptide chain synthesis: an A residue at this position permits the UGA readthrough process.

Base Sequence↗
PubMed1981

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.

Bacterial Proteins↗