The assembly of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins) into ribosomal subunits (30S and 50S) is a complex process. Transcription of rRNA requires antitermination proteins and the primary transcripts are processed by ribonucleases. R-proteins and rRNAs are chemically modified, the r-proteins bind to the rRNAs and the formed RNA-protein complexes are folded into mature ribosomal subunits. All these processes are well-coordinated and overlapping. Non-ribosomal factors are required for proper assembly and maturation of the ribosomal subunits. Two of these factors are the RimM and RbfA proteins, which bind to 30S subunits and are important for efficient processing of 16S rRNA. Lack of either RimM or RbfA results in a reduced amount of polysomes and a lower growth rate. An increased amount of RbfA can partially compensate for deficiencies shown by a RimM lacking mutant. Here, mutations that alter phylogenetically conserved amino acids in RimM have been constructed. One of these (rimM120), which resulted in the replacement of two adjacent tyrosines by alanines, reduced the growth rate three-fold and also decreased the processing efficiency of 16S rRNA. The RimM120 mutant protein showed a much reduced binding to the 30S subunits. Suppression of the rimM120 mutant was achieved by increased amount of the RimM120 protein, by overexpression of rbfA, or by mutations that changed r-protein S19 or 16S rRNA. A variant of r-protein S13, which was previously isolated as a suppressor to a deletion of rimM (∆rimM), suppressed also the rimM120 mutation. The wild-type RimM protein, but not the RimM120 protein, was shown to bind r-protein S19 in the 30S subunits. The changes in S13, S19 and 16S rRNA that compensated for the deficiencies shown by the rimM mutants are all located within a small region of the head of the 30S subunit, suggesting that this region is the likely target for the RimM action. To isolate RbfA variants that show reduced association with the 30S subunits, phylogenetically conserved, surface exposed amino acid residues of RbfA were changed to alanines or, in some instances, to amino acids of the opposite charge to that in the wild-type protein. Alterations of F5, R31, D46 and D100 had the largest effect on growth. Mutations in the metY-nusA-infB operon, isolated as suppressors to the ∆rimM mutant, were shown to increase the amounts of RbfA. In a ∆rimM mutant, all RbfA protein was found associated with the 30S subunits and no free RbfA was detected. The RlmB protein was shown to be the methyltransferase responsible for the formation of Gm2251 in 23S rRNA in Escherichia coli. Unlike a Saccharomyces cerevisiae mutant that lacks the orthologue to RlmB, Pet56p, which methylates mitochondrial rRNA, a ∆rlmB mutant did not show any defects in ribosomal assembly.

<p>The ribosome is part of the indispensable machinery of every living cell. This large macromolecule, which decodes messenger RNA to produce proteins, is the subject of intense study as the mediator of an essential process. The prokaryotic ribosome is a major target for antimicrobial therapy, as its structure differs significantly from the eukaryotic ribosome. At present, the in vivo process of translation on the mature bacterial, or 70S, ribosome is well studied and increasingly understood, while the process of assembling the small (30S) and large (50S) subunits of this complex ribonucleoprotein enzyme has mostly been studied in vitro. Consequently, the significance of in vivo events such as ribosomal RNA (rRNA) maturation and factor-mediated maturation is incompletely understood. By studying the nature and structure of an in vivo assembled immature 30S subunit, this thesis aims to gain a better understanding of the key events in 30S subunit biogenesis. Deletion of the assembly cofactor Ribosome Maturation Factor M (RimM) results in slow growth, inefficient rRNA processing, and accumulation of nonfunctional, immature 30S subunits. This work presents the first cryo-EM model of the immature 30S purified from a RimM knockout strain of <em>E. coli</em>. The structure reveals distortion of the decoding centre and a disrupted 50S-binding interface, attesting to the importance of rRNA processing in 30S maturation. Additionally, the model suggests consequences for ribosomal protein incorporation and rRNA domain position relative to the mature 30S.</p> / Master of Science (MSc)