Growth rate regulation of translation initiation factor IF3 biosynthesis in Escherichia coli.

infC, the gene encoding translation initiation factor IF3 in Escherichia coli, can be transcribed from three promoters. Two of these promoters, PI1 and PI2, are located in the upstream thrS sequence which codes for threonyl-tRNA synthetase. Previous studies had shown that PI2 was the major promoter for infC. In the present study, the extent of transcription from PI1 and/or PI2 at a variety of steady-state growth rates was analyzed by promoter fusion studies. PI2 was the more active promoter (two- to threefold stronger than PI1) at all growth rates tested. A fusion plasmid containing both PI1 and PI2 exhibited a transcription level approximately equal to the sum of those observed with the fusion plasmids containing the individual promoters. The transcriptional activities of PI1 and PI2 did not change as the growth rate was varied from 0.3 to 1.7 doublings per h. In contrast, a fusion plasmid carrying the rrnB P1 promoter displayed the expected growth rate response. The steady-state concentrations of infC mRNA in cells grown at different rates were measured and found not to vary. These results indicate that the previously reported growth rate regulation of IF3 biosynthesis neither is accomplished by transcriptional control nor is a result of differential mRNA stability. In view of these results, the steady-state levels of IF3 in cells grown at a number of different growth rates were determined by quantitative immunoblotting. IF3 levels were found to vary with growth rate in a manner essentially identical to that observed for ribosomes. A model accounting for these results and describing a mechanism for coordinate growth rate-regulated expression of ribosomes and IF3 is presented.

Transcriptional patterns for the thrS-infC-rplT operon of Escherichia coli.

The genes coding for threonyl-tRNA synthetase (thrS), translation initiation factor 3 (infC) and ribosomal protein L20 (rplT) are clustered in the Escherichia coli genome. Previous studies had suggested the possibility that the expression of these genes is coupled. The transcriptional events in this operon have now been examined by S1 nuclease mapping and promoter fusion studies. The results indicate that infC-containing mRNAs are initiated from three separate promoters. Two of these are located in the protein-coding region of thrS and one, P12, is the major promoter at all growth rates tested. In addition, there is co-transcription of thrS and infC from the thrS promoter (PT). A single promoter for thrS has been mapped approx. 170 nucleotides upstream from its translation initiation site. Another promoter has been located within the infC-coding region. It is separated from the next downstream gene, rplT, by a transcription end point. However, termination at this region is only 50-70% efficient and transcripts starting at this promoter can read through into rplT. These findings demonstrate that the pattern of transcription in this operon is highly complex and the mRNA levels for each of the genes is determined by a variety of factors, including multiple promoters, co-transcription and readthrough of transcription termination signals.

Measurement of cat expression from growth-rate-regulated promoters employing beta-lactamase activity as an indicator of plasmid copy number.

Many promoter-fusion vectors contain an intact beta-lactamase (BLA) gene (bla) to allow measurement of BLA activity as an internal control for plasmid copy number. This approach rests on the assumption that bla is constitutively expressed. To use such vectors for comparison of promoter activity at different growth rates it was necessary to confirm that this is the case under all physiological conditions. The relationship between plasmid copy number and BLA activity at different steady-state growth rates in Escherichia coli HB101 transformed with a ColE1-type plasmid (pBR325) was examined. Both BLA activity and plasmid copy number decreased in a parallel fashion as growth rate increased. This finding was tested further by measuring the growth-rate-regulated expression of the chloramphenicol acetyltransferase (CAT) gene under the control of the rrnB P1 promoter in a plasmid pKK231-1 fusion. The results indicate that BLA activity is a reliable indicator of copy number at a wide range of growth rates and that CAT/BLA ratios can be employed as a valid measure of promoter-specific activity in such plasmid fusions under these different physiological conditions.

Molecular cloning and sequencing of pheU, a gene for Escherichia coli tRNAPhe.

A recombinant plasmid (designated pID2) carrying the E. coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E. coli K12 DNA into the EcoRI site of pACYC184 DNA. The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase. Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity. The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2. The sequence of the gene and its flanking regions is presented.