N. Glansdorff

Heterotropic interactions in Escherichia coli aspartate transcarbamylase : subunit interfaces involved in CTP inhibition and ATP activation

In Escherichia coli aspartate transcarbamylase, each regulatory chain is involved in two kinds of interfaces with the catalytic chains, one with the neighbour catalytic chain which belongs to the same half of the molecule (R1-C1 type of interaction), the other one with a catalytic chain belonging to the other half of the molecule (R1-C4 type of interaction). In the present work, site-directed mutagenesis was used to investigate the involvement of the C-terminal region of the regulatory chain in the process of feed-back inhibition by CTP. Removal of the two last C-terminal residues of the regulatory chains is sufficient to abolish entirely the sensitivity of the enzyme to CTP. Thus, it appears that the contact between this region and the 240s loop of the catalytic chain (R1-C4 type of interaction) is essential for the transmission of the regulatory signal which results from CTP binding to the regulatory site. None of the modifications made in the R1-C4 interface altered the sensitivity of the enzyme to the activator ATP, suggesting that the effect of this nucleotide rather involves the R1-C1 type of interface. These results are in agreement with the previously proposed interpretation that CTP and ATP do not simply act in inverse ways on the same equilibrium.

Homologous control sites and DNA transcription starts in the related argF and argI genes of Escherichia coli K12.

The argF and argI genes code for similar proteins able to assemble into hybrid isoenzymes and are therefore thought to share a common origin. We show here that the nucleotide sequence of the promoter and operator regions of these two genes are highly homologous. DNA regions preceding the control sites also present significant homologies. The results support the notion of divergent evolution of the two genes from a common ancestor. Like argE and argCBH, argF and argI are controlled by a repressor molecule recognizing a family of similar operator sites. Attenuation appears to play no role in this regulation.

On the role of the Shine-Dalgarno sequence in determining the efficiency of translation initiation at a weak start codon in the car operon of Escherichia coli K12☆

Translation of the carA gene is efficiently initiated at the intrinsically weak UUG codon. A single nucleotide substitution changing the Shine-Dalgarno box of carA (GGAGG) into the sequence TGAGG reduces translation of carA sevenfold. This result supports the view that extensive complementarity between the Shine-Dalgarno sequence and 16S RNA contributes significantly to the efficiency of translation when the latter starts at a weak initiation triplet.

Enhancement of translation efficiency in Escherichia coli by mutations in a proximal domain of messenger RNA

Abstract The effect of two mutations in Escherichia coli located 5′ to the Shine-Dalgarno sequence of argE messenger RNA suggests that maximal translation efficiency may depend on the composition of proximal sequences not necessarily involved in secondary structures.

The promoter region of the arg3 gene in Saccharomyces cerevisiae: nucleotide sequence and regulation in an arg3-lacZ gene fusion.

We have determined the DNA sequence for the 5′ end of the arg3 gene of Saccharomyces cerevisiae, including part of the coding region and the 200 nucleotides immediately upstream. A promoter‐deletion mutant was found to have lost all of the sequence lying normally in front of the gene except for the 33 nucleotides preceding the AUG codon. The role of the 5′ domain in initiation and regulation of arg3 transcription was assessed by a gene fusion experiment. The Escherichia coli lacZ gene, was truncated of the eight amino‐terminal codons substituted in vitro, on a 2mu plasmid, for the carboxy‐terminal and 3′‐flanking regions of arg3, leaving only the first 19 proximal codons and approximately 1600 nucleotides of the region preceding arg3 on the yeast chromosome. The fused gene was expressed in phase and was still submitted to the two mechanisms regulating the wild‐type arg3 gene: the general, probably transcriptional control of amino acid biosynthesis and the specific, apparently post‐transcriptional control mediated by the products of the argR genes. These results suggest a determining role for the 5′ end portion of the arg3 messenger in the specific arginine‐mediated control mechanism.

On the role of the Escherichia coli integration host factor (IHF) in repression at a distance of the pyrimidine specific promoter P1 of the carAB operon

Binding of integration host factor to its target site, centered around nucleotide -305 upstream of the transcription startpoint, exerts antagonistic effects on the expression of P1, the upstream pyrimidine specific promoter of the E coli and S typhimurium carAB operons. IHF stimulates P1 promoter activity in minimal medium, but also increases the repressibility of this promoter by pyrimidines. We present evidence strongly suggesting that IHF exerts these effects by modulating the binding of another pyrimidine specific regulatory molecule, probably the product of gene carP. The carAB control region contains a GATC Dam methylation site, 106 bp upstream of the P1 transcription startpoint, which can be protected in vivo against methylation. This protection requires at least the regulatory carP gene product and a high pyrimidine nucleotide pool and, as shown here, the integration host factor. Whether CarP directly binds to this site or exerts its protective effect indirectly is not yet known. In the absence of IHF (himA) or in mutants affected in the IHF target site this protection is strongly impaired, suggesting that IHF positively influences the formation or the stability of the protective protein-DNA complex some 200 bp downstream. Furthermore, we have demonstrated that the distance separating the IHF and GATC Dam methylase target sites is crucial for the in vivo protection and for pyrimidine mediated regulation of P1 promoter expression. Indeed, shortening this distance by 6 bp, and more surprisingly also by 11 bp, results in a severe reduction of the degree of in vivo protection of the GATC site against methylation and concomitantly of the repressibility by pyrimidines of P1 promoter activity. The absence of both these effects in a double, deletion-duplication, mutant resulting in a net increase of the intervening sequence by 1 bp, clearly demonstrates that these effects are not due to the disruption of an important regulatory site, but must be attributed to variations in the distance separating different protein binding sites.

Tandem and inverted repeats of arginine genes in Escherichia coli

SummaryDuplications of arg genes produced in the Rec+ and in the recA genetic backgrounds are shown by heteroduplex analysis to be strictly tandem at the level of resolution of this technique. The formation of these particular rearrangements therefore does not require the inclusion of transposons or other sequences of an appreciable size in their final structure.Duplications of short segments (about 2,000 nucleotides) appear unexpectedly stable when compared with duplications of longer segments (about 10,000 nucleotides).One of the structures analyzed displays two inversely repeated argE genes rearranged into an artificial divergent operon. The bearing of this observation on the origin of bipolar operons, of “mirror-image” map symmetries and on the production of inverted repeats in general, is discussed.

Studies on the control region of the bipolar argECBH operon of Escherichia coli

SummarySeveral mutations affecting the control or the potential of gene expression in the argECBH bipolar operon have been characterized by enzyme assays, genetic mapping, dominance tests and pulse labelled RNA determinations. None of the mutations involves DNA rearrangements detectable by heteroduplex analysis (Charlier et al., 1978).Partially constitutive transcription of both argE and argCBH has been observed in mutant L10 while constitutive argE transcription and normal argCBH control characterize mutants L9, LL13 and LL2. The control region thus appears to contain two overlapping operators, as suggested previously (Elseviers et al., 1972).Two mutants (L2, LL1) and strain 6–8 from Bretscher and Baumberg (1976) display an increase in acetylornithinase specific activity (argE product) without concommittant increased argE transcription. In addition, they exhibit a decreased argCBH transcription. It is suggested that in these organisms, argE translation and argCBH transcription may be affected by the same genetic event; this explanation is compatible with the present working hypothesis for the structure of the control region. An interpretation in terms of messenger attenuation also appears possible.From the properties of two strains harbouring an IS2 insertion in the control region (Charlier et al., 1978) the following conclusion may be drawn:1.When inserted in orientation I close to the proximal end of a silent gene IS2 appears to promote a low but detectable transcription readthrough into that gene.2.Insertion of an IS2 element in orientation II close to a neighbouring gene is not a sufficient condition to express that gene at a high rate.