Browsing by Subject "ribosomal protein"
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Item Domain I of 23S rRNA competes with a paused transcription complex for ribosomal protein L4 of Escherichia coli(Oxford Universiiy Press, 1993-05-25) Zengel, J. M.; Lindahl, L.Ribosomal protein L4 of Escherichia coli regulates expression of its own eleven gene S10 operon both by inhibiting translation and by stimulating premature termination of transcription. Both regulatory processes presumably involve L4 recognition of the S10 leader RNA. To help define L4's regulatory target, we have investigated the protein's cognate target on 23S rRNA. Binding of L4 to various fragments of the 23S rRNA was monitored by determining their ability to sequester L4 in an in vitro transcription system and thereby eliminate the protein's effect on transcription. Using this approach we identified a region of about 110 bases within domain I of 23S rRNA which binds L4. A two base deletion within this region, close to the base to which L4 has been cross-linked in intact 50S subunits, eliminates L4 binding. These results also confirm the prediction of the autogenous control model, that L4 bound to its target on rRNA is not active in regulating transcription of the S10 operon.Item A hairpin structure upstream of the terminator hairpin required for ribosomal protein L4-mediated attenuation control of the S10 operon of Escherichia coli(American Society for Microbiology, 1996-04) Zengel, J. M.; Lindahl, L.Ribosomal protein L4 of Escherichia coli regulates transcription of the 11-gene S1O operon by promoting premature termination of transcription (attenuation) at a specific site within the 172-base untranslated leader. We have analyzed the roles of various domains of the leader RNA in this transcription control. Our results indicate that the first 60 bases of the leader, forming the three proximal hairpin structures, are not essential for in vivo L4-mediated attenuation control. However, a deletion removing the fourth hairpin, which is immediately upstream of the terminator hairpin, eliminates L4's effect on transcription. Base changes disrupting complementarity in the 6-bp stem of this hairpin also abolish L4 control, but compensatory base changes that restore complementarity also restore L4's effect. In vitro transcription studies confirm that this hairpin structure is necessary for L4's role in stimulating transcription termination by RNA polymerase.Item Phylogenetic Analysis of L4-Mediated Autogenous Control of the S10 Ribosomal Protein Operon(American Society for Microbiology, 1999-07-25) Allen, Todd; Shen, Ping; Samsel, Leigh; Liu, Raymond; Lindahl, Lasse; Zengel, Janice M.We investigated the regulation of the S10 ribosomal protein (r-protein) operon among members of the gamma subdivision of the proteobacteria, which includes Escherichia coli. In E. coli, this 11-gene operon is autogenously controlled by r-protein L4. This regulation requires specific determinants within the untranslated leader of the mRNA. Secondary structure analysis of the S10 leaders of five enterobacteria (Salmonella typhimurium, Citrobacter freundii, Yersinia enterocolitica, Serratia marcescens, and Morganella morganii) and two nonenteric members of the gamma subdivision (Haemophilus influenzae and Vibrio cholerae) shows that these foreign leaders share significant structural homology with the E. coli leader, particularly in the region which is critical for L4-mediated autogenous control in E. coli. Moreover, these heterologous leaders produce a regulatory response to L4 oversynthesis in E. coli. Our results suggest that an E. coli-like L4-mediated regulatory mechanism may operate in all of these species. However, the mechanism is not universally conserved among the gamma subdivision members, since at least one, Pseudomonas aeruginosa, does not contain the required S10 leader features, and its leader cannot provide the signals for regulation by L4 in E. coli. We speculate that L4-mediated autogenous control developed during the evolution of the gamma branch of proteobacteria.Item Regulation of Ribosomal Protein Synthesis in Vibrio cholerae(American Society for Microbiology, 2004-09) Allen, Todd D.; Watkins, Tonya; Lindahl, Lasse; Zengel, Janice M.We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria.Item Surprising flexibility of leader RNA determinants for r-protein L4-mediated transcription termination in the Escherichia coil S10 operon(The RNA Society, 2002-05) Zengel, Janice M.; Sha, Yizhong; Lindahl, LasseEscherichia coli ribosomal protein L4 autogenously regulates transcription of the S10 operon, which encodes L4 and 10 other ribosomal proteins. Regulation results from L4-stimulated premature transcription termination at a U-rich site in the untranslated leader. The process requires transcription factor NusA. Here we report a detailed analysis of the RNA requirements for NusA-dependent, L4-mediated transcription control. We found that efficient regulation requires multiple features of the S10 leader, including two hairpins, called HD and upper HE, a connecting tether, and a U-rich sequence at the distal side of HE. As expected, regulation was optimal when all 7 Us were maintained in the U4CGU3 sequence at the termination site. However, despite the apparent specificity of L4 action on only the S10 operon, there is surprising flexibility at the primary sequence level for the HD-tether-HE region. Changes in the sequence of non-base-paired nucleotides flanking the HD hairpin or an A at the second position of the HD loop reduced L4 regulation, but other changes had little or no effect. Furthermore, generic hairpins from other RNAs could replace the natural HD and upper HE hairpins with little or no reduction of L4 control, suggesting that the secondary structure elements are also relatively generic. The lack of specific sequence requirements suggests that L4 may recognize multiple elements within this region of the nascent leader.