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Abstract
The coordinated synthesis and assembly of ribosomal subunits and ribosomal RNA (rRNA) in Escherichia coli is a highly complex and well-regulated process. One aspect of this control that is often overlooked is the post-transcriptional regulation of rRNA. The work described in this dissertation analyzes the processing of rRNAs in E. coli. In E. coli all the three rRNAs are co-transcribed as 30S primary transcript from seven rRNA operons which is initially cleaved by RNase III to generate pre-rRNA species and are subsequently processed by various endo- and exoribonucleases to generate the mature ends. Here we show that the 30S primary transcript is efficiently processed by RNase E, RNase G and YbeY in absence of RNase III. This alternative processing pathway which works in absence of RNase III is a backup mechanism for cells to process 30S rRNAs. Moreover, the alternative processing pathway generates multiple cleavage sites which are unique from the RNase III-dependent pathway. We also found that after the initial cleavages of 30S rRNA, the pre-16S and pre-23S rRNA species are separated from each other and are subsequently processed by the secondary processing enzymes-RNase P, RNase Z and exoribonucleases to generate the mature rRNA species. We have also identified three new genes (YhgF, YraN and YhbQ) with possible enzymatic activity which play a role in the processing of pre-23S rRNA in the alternative processing pathway. Here we also analyzed the alternative processing pathway in the individual rRNA operons and showed that the heterogeneities seen in the gene organization among the seven rRNA operons influence the 30S rRNA processing pattern. The primary processing enzymes of the alternative processing pathway has different efficiencies in rrnD and rrnG rRNA operon. In fact, we also showed that in the alternative processing pathway, the 5 ends of some of the cleavage products in rrnD rRNA operon are not the same as in rrnG operon. Additionally, we also showed that the presence of the spacer tRNA in between the 16S and 23S rRNA is essential for 30S rRNA processing in absence of RNase III. While this work is not complete yet, we have constructed the tools required for the analysis of significance of spacer tRNA in rRNA processing in E. coli