Analysis of sequence elements important for the synthesis and control of ribosomal RNA in E coli

Abstract

The regulation of the synthesis of ribosomal RNA is a key problem for the understanding of bacterial growth. Many different regulatory mechanisms involving cis and trans acting components participate in a concerted way to achieve the very efficient, flexible and coordinated production of this class of molecules. We have studied three different sequence regions within a ribosomal RNA transcription unit which are believed to control different stages of ribosomal RNA expression. In the first part of the study the function of AT-rich sequences upstream of the -35 hexamer of rRNA promoter P1 in the activation of rRNA transcription was analyzed. We confirm that a sequence dependent bend upstream of P1 is responsible for the high promoter activity. Experiments employing linker scanning mutations demonstrated that the distance as well as the angular orientation of the bent DNA is crucial for the degree of activation. In addition, the effect of the trans activating protein Fis on the transcription initiation of promoter P1 was investigated. We can show, using the abortive initiation assay, that the predominant effect of Fis is due to an increase in the affinity of RNA polymerase for the promoter (binding constant KB) while the isomerisation rate (kf) from a closed to an open RNA polymerase promoter complex is not altered significantly. We also describe the characterization of sequence determinants important for stringent regulation and growth rate control. Evidence is provided that the discriminator motif GCGC is a necessary but not sufficient element for both types of control. Furthermore we show that not simply a particular DNA primary structure but the higher order conformation of the complete promoter region is recognized and triggers the two regulatory mechanisms, both of which are apparently mediated by the effector molecule guanosine tetraphosphate (ppGpp). Finally, we have carried out a systematic mutational analysis of the rrnB leader region preceding the structural gene for 16S RNA. We could demonstrate that highly conserved sequence elements within the rrnB leader, which were believed to be involved in transcription antitermination have post-transcriptional functions. We present evidence that these sequence elements direct the biogenesis of active ribosomal particles.