Effects of Fis on ribosome synthesis and activity and on rRNA promoter activities in Escherichia coli.
The DNA-bending protein Fis of Escherichia coli is required for efficient initiation of chromosome replication and for activation of the P1 promoters of stable RNA (rRNA and tRNA) genes. Using fis+ and fis- bacterial strains, we have determined ribosome synthesis and activity, rRNA gene dosage, and transcript initiation rates at the rrnB P1 and P2 promoters. Different growth media were used to achieve growth rates between 0.7 and 2.7 doublings/hour for the fis+ strain, and between 0.7 and 2.2 doublings/hour for the fis- strain. In minimal media, the Fis deficiency only reduced the DNA and rrn gene concentration (DNA, oriC copies and rrn genes per protein); in amino acid-supplemented media, it also reduced rRNA synthesis rates per protein. Under all conditions, the ribosome activity (protein synthesis rate/ribosome) remained unchanged by the absence of Fis. In the presence of Fis, the absolute activities of the isolated rrnB P1 and P2 promoters increased from 2 to 88 and 10 to 50 initiations/minute, respectively, with increasing growth-rate. In the absence of Fis, these activities increased from 3 to 70 and 10 to 80 initiations/minute, respectively. Relative to the isolated rrnB P2 promoter, the strength of the rrnB P1 promoter was found to increase with increasing growth rate tenfold (from 0.17 to 1.7) in the presence, but only fivefold (from 0.17 to 0.85) in the absence of Fis. An evaluation of the data leads us to propose estimates of kcat and relative KM values for the two rRNA promoters, and relative values for free RNA polymerase concentrations during growth in different media. The analysis suggests that the reduced strength of P1 promoters of stable RNA genes in the absence of Fis, together with the reduced rrn gene concentration, increases the concentration of free RNA polymerase. In addition, the lower rrn P1 promoter activity in the absence of Fis reduces the probability that the downstream P2 promoter is blocked ("occluded") by a transcription elongation complex originating at the P1 promoter. The increased polymerase concentration and reduced P2 promoter occlusion both help to compensate for the Fis-deficiency, but during growth in rich media when the demand for ribosomes is high, this compensation is insufficient for fis- bacteria to achieve the wild-type level of ribosome synthesis and growth.
Zhang
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Molecular Program, University of Texas at Dallas, Richardson 75083-0688, USA.
Bremer
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D002352:Carrier Proteins / Q000235:genetics*; D003470:Culture Media; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D015964:Gene Expression Regulation, Bacterial; D034982:Integration Host Factors; D011401:Promoter Regions, Genetic; D012335:RNA, Ribosomal / Q000096:biosynthesis* / Q000235:genetics; D018741:Replication Origin; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D012335:RNA, Ribosomal; C440502:integration host factor, E coli; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase