Maria Stella Carlomagno

Structure and function of the Salmonella typhimurium and Escherichia coli K-12 histidine operons.

We have determined the complete nucleotide sequence of the histidine operons of Escherichia coli and of Salmonella typhimurium. This structural information enabled us to investigate the expression and organization of the histidine operon. The proteins coded by each of the putative histidine cistrons were identified by subcloning appropriate DNA fragments and by analyzing the polypeptides synthesized in minicells. A structural comparison of the gene products was performed. The histidine messenger RNA molecules produced in vivo and the internal transcription initiation sites were identified by Northern blot analysis and S1 nuclease mapping. A comparative analysis of the different transcriptional and translational control elements within the two operons reveals a remarkable preservation for most of them except for the intercistronic region between the first (hisG) and second (hisD) structural genes and for the rho-independent terminator of transcription at the end of the operon. Overall, the operon structure is very compact and its expression appears to be regulated at several levels.

Control of mRNA processing and decay in prokaryotes

Post-transcriptional mechanisms operate in regulation of gene expression in bacteria, the amount of a given gene product being also dependent on the inactivation rate of its own message. Moreover, segmental differences in mRNA stability of polycistronic transcripts may be responsible for differential expression of genes clustered in operons. Given the absence of 5′ to 3′ exoribonucleolytic activities in prokaryotes, both endoribonucleases and 3′ to 5′ exoribonucleases are involved in chemical decay of mRNA. As the 3′ to 5′ exoribonucleolytic activities are readily blocked by stem-loop structures which are usual at the 3′ ends of bacterial messages, the rate of decay is primarily determined by the rate of the first endonucleolytic cleavage within the transcripts, after which the resulting mRNA intermediates are degraded by the 3′ to 5′ exoribonucleases. Consequently, the stability of a given transcript is determined by the accessibility of suitable target sites to endonucleolytic activities. A considerable number of bacterial messages decay with a net 5′ to 3′ directionality. Two different alternative models have been proposed to explain such a finding, the first invoking the presence of functional coupling between degradation and the movement of the ribosomes along the transcripts, the second one implying the existence of a 5′ to 3′ processive ‘5′ binding nuclease’. The different systems by which these two current models of mRNA decay have been tested will be presented with particular emphasis on polycistronic transcripts.

The histidine operon of Azospirillum brasilense: organization, nucleotide sequence and functional analysis

A 3457-base pair fragment of Azospirillum brasilense DNA which complemented mutations in the hisA and hisF genes of Escherichia coli was sequenced. The sequence analysis revealed the presence of six major contiguous open reading frames (ORF). The comparison of the predicted amino acid sequence of these ORF with those encoded by the eubacterial, archaebacterial and eukaryotic his genes sequenced thus far revealed that four of them have a significant degree of homology with the E. coli hisH, hisA, hisF and the C-terminal domain of the hisI gene products. S1 mapping experiments indicated that the putative transcription start site coincided with the AUG translational initiation codon of the hisBd gene, the first gene of the A. brasilense his operon. Downstream from the last ORF, a sequence was identified which functions as a Rho-independent transcription terminator. Comparison of amino acid sequences, gene order and organization and evolutionary aspects of the A. brasilense his cluster are discussed.

Processing of a polycistronic mRNA requires a 5′cis element and active translation

We have characterized a major processed species of mRNA in the his operon of Salmonella typhimurium. In vivo and in vitro analyses of the his transcripts from wild‐type and mutant strains using S1 nuclease protection assays, measurements of RNA stability, deletion mapping, gel retardation, and in vitro translation assays demonstrate that the distal portion of the polycistronic his mRNA is processed, resulting in increased stability. The processing event requires an upstream cis‐acting element and translation of the cistron immediately downstream of the 5′ end of the processed species. The cistrons contained in this segment are also independently transcribed from an internal promoter which is maximally active in the absence of readthrough transcription from the primary promoter.

A cytosine‐ over guanosine‐rich sequence in RNA activates rho‐dependent transcription termination

We have constructed an expression vector carrying the Escherichia coli his operon control region to study the ability of defined segments of DNA to cause rho tactor‐mediated transcription termination both in vivo and in vitro. We have previously identified a consensus motif consisting of a region of high cytosine over guanosine content common to several cryptic intracistronic transcription termination elements unmasked by polar mutations. We show that a DNA fragment possessing features similar to the ones previously identified is capable of causing rho‐mediated release of transcripts in vivo and in vitro. The efficiency of termination depends on the length and relative cytosine over guanosine ratio of the element.

Gene structure in the histidine operon of Escherichia coli

SummaryThe bifunctional enzyme imidazoleglycerolphosphate dehydratase and histidinolphosphate phosphatase is encoded by the hisB gene. The fourth gene of the histidine operon, hisB, was cloned and mapped on a 2,300 base pair DNA fragment. In the present study we report the complete nucleotide sequence of the hisB gene of Escherichia coli. The gene is 1,068 nucleotides long and codes for a protein of 355 amino acids with an apparent molecular weight of 39,998 daltons. The protein product(s) of the hisB region of both Salmonella typhimurium and E. coli were identified by subcloning and expression in an in vitro translation system. In both organisms the hisB gene directed the synthesis of a single protein with an apparent molecular weight of 40,500 daltons, consistent with the data derived from the nucleotide sequence analysis.

Gene organization in the distal part of the Salmonella typhimurium histidine operon and determination and sequence of the operon transcription terminator

SummarySeveral transducing phages, carrying different deletions of the Salmonella thyphimurium histidine operon were constructed and mapped. These phages were used to obtain fragments of DNA comprising different regions of the operon, which were subcloned in plasmid vectors. The recombinant plasmids allowed the construction of a physical and restriction map of the histidine operon. The presence of the different genes on individual fragments was confirmed by complementation tests. The transcription termination site of the histidine operon has been established by S1 mapping and sequence analysis. The entire operon measures about 7100 base pairs and the last six structural genes are contained in 3450 bases of genetic material.