Browsing by Subject "ribosomal protein operon"
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Item Autogenous control is not sufficient to ensure steady-state growth rate-dependent regulation of the S10 ribosomal protein operon of Escherichia coli(American Society for Microbiology, 1990-01) Lindahl, L.; Zengel, J. M.The regulation of the S10 ribosomal protein operon of Escherichia coli was studied by using a lambda prophage containing the beginning of the S10 operon (including the promoter, leader, and first one and one-half structural genes) fused to lacZ. The synthesis of the lacZ fusion protein encoded by the phage showed the expected inhibition during oversynthesis of ribosomal protein L4, the autogenous regulatory protein of the S10 operon. Moreover, the fusion gene responded to a nutritional shift-up in the same way that genuine ribosomal protein genes did. However, the gene did not exhibit the expected growth rate-dependent regulation during steady-state growth. Thus, the genetic information carried on the prophage is sufficient for L4-mediated autogenous control and a normal nutritional shift-up response but is not sufficient for steady-state growth rate-dependent control. These results suggest that, at least for the 11-gene S10 ribosomal protein operon, additional regulatory processes are required to coordinate the synthesis of ribosomal proteins with cell growth rate and, furthermore, that sequences downstream of the proximal one and one-half genes of the operon are involved in this control.Item Autogenous control of the S10 ribosomal protein operon of Escherichia coli: genetic dissection of transcriptional and posttranscriptional regulation(National Academy of Sciences, 1987-09) Freedman, L. P.; Zengel, J. M.; Archer, R. H.; Lindahl, L.The S10 ribosomal protein operon is regulated autogenously by the product of one of the genes of the operon, the gene encoding ribosomal protein L4. We have used site-directed mutagenesis to isolate leader mutations affecting L4 control. The phenotypes of these mutants demonstrate that L4 regulates both transcription and translation of the S10 operon. Several mutations abolish both levels of L4 control; others eliminate either transcriptional or translational control with little or no effect on the other mode of regulation. We conclude that L4-mediated transcriptional and translational control share some sequence requirements, but the two regulatory processes recognize somewhat different features of the S10 leader. Primary as well as secondary structures within the S10 leader appear to be involved.Item Ribosomal protein L4 stimulates in vitro termination of transcription at a NusA-dependent terminator in the S10 operon leader(National Academy of Sciences, 1990-04) Zengel, J. M.; Lindahl, L.The 11-gene S10 ribosomal protein operon of Escherichia coli is under the autogenous control of L4, the product of the third gene of the operon. Ribosomal protein L4 inhibits both transcription and translation of the operon. Our in vivo studies indicated that L4 regulates transcription by causing premature termination within the untranslated S10 operon leader. We have now used an in vitro transcription system to study the effect of purified L4 on expression of the S10 operon. We find that the cell-free system reproduces the in vivo observations. Namely, in the absence of L4, most of the RNA polymerases read through the termination site in the S10 attenuator; the addition of L4 results in increased termination at this site. However, RNA polymerase does not terminate at the S10 attenuator, with or without L4, unless an additional factor, protein NusA, is added to the transcription reaction. These results suggest that the attenuator in the S10 operon is a NusA-dependent terminator whose efficiency is regulated by ribosomal protein L4.Item Secondary structure of the leader transcript from the Escherichia coli S10 ribosomal protein operon(Oxford University Press, 1988-09-26) Shen, P.; Zengel, J. M.; Lindahl, L.Genetic analysis of the autogenous control of the S10 ribosomal protein operon of Escherichia coli has suggested that the secondary or tertiary structure of the leader transcript is important for this regulation. We have therefore determined the secondary structure of the leader by enzyme digestion and chemical modification. Our results suggest that the 172 base leader exists in two forms, differing only immediately upstream of the Shine-Dalgarno sequence of the first gene. We discuss the possibility that the equilibrium between these alternate structures is important for the L4-mediated regulation of translation of the S10 operon. We have also determined the structure of several mutant transcripts. Correlation of these structures with the regulatory phenotypes suggest that a hairpin about 50 bases upstream of the first gene is essential for the control of translation of the operon. Finally, our results show that a two base substitution in an eight base loop destabilizes the attached stem.Item Translational coupling of the two proximal genes in the S10 ribosomal protein operon of Escherichia coli(American Society for Microbiology, 1989-05) Lindahl, L.; Archer, R. H.; McCormick, J. R.; Freedman, L. P.; Zengel, J. M.We have examined the translational coupling between the first two genes in the S10 ribosomal protein operon. We isolated mutations blocking the translation of the first gene of the operon, coding for S10, and monitored their effects on translation of the downstream gene, coding for L3. All of the mutations inhibiting S10 synthesis also affected the synthesis of L3. However, these experiments were complicated by decreased mRNA synthesis resulting from transcription polarity, which we could only partially eliminate by using a rho-100 strain. To completely eliminate the problem of transcription polarity and obtain a more accurate measurement of the coupling, we replaced the natural S10 promoter with a promoter used by the bacteriophage T7 RNA polymerase. As expected, the T7 RNA polymerase was not subject to transcription polarity. Using this system, we were able to show that a complete abolishment of S10 translation resulted in an 80% inhibition of L3 synthesis. Other experiments show that the synthesis of L3 goes up as a function of increasing S10 synthesis, but the translational coupling does not assure strictly proportional output from the two genes.