The importance of maturation factors in 30S ribosomal subunit assembly

Sammanfattning: The assembly of the ribosome is a complex process that needs to be highly efficient to support maximum growth. Although the individual subunits of the ribosome can be reconstituted in vitro, such a reaction is inefficient in comparison to the assembly rate in vivo. What differentiates the in vivo from the in vitro assembly is primarily the presence of ribosome assembly proteins. These are proteins that assist in the assembly of the ribosomal subunits but are not part of the mature ribosome. In bacteria, the ribosome assembly proteins include rRNA processing enzymes and rRNA/ribosomal protein (r-protein) modifying enzymes. One set of ribosome assembly proteins, the ribosome maturation factors, have been difficult to classify due to their differences in structure and their apparent lack of similarities with regard to function. As part of this thesis, the previously uncharacterized RimP (ribosome maturation) protein formerly known as P15A or YhbC, was studied. Deletion of the rimP gene affected the growth rate more severely at 44°C than at 37°C and 30°C. Polysome profile analysis revealed a decrease in the amount of translating ribosomes and a corresponding increase in the amount of free 50S and 30S ribosomal subunits. The disproportionate large increase in 50S relative to 30S subunits indicated a 30S assembly defect. RimP was shown to localize to the 30S ribosomal subunit, and an accumulation of 17S rRNA, a precursor to 16S rRNA, supports a role for RimP in 30S subunit maturation. The results from in vitro reconstitution experiments have given valuable insights in the assembly of the 30S subunit. By using a recently developed method, the role of ribosome maturation factors Era, RimM and RimP during in vitro reconstitutions of the 30S subunit was investigated. Era was found to increase the incorporation rate for most of the late binding r-proteins, while RimM and RimP had more specific effects. RimM increased the incorporation rate for r-proteins S19 and S9 and inhibited the incorporation of S13 and S12, whereas RimP increased the incorporation rate primarily for S12 and S5. A comparison of the ribosome maturation factors RimP and RbfA (ribosome binding factor A) revealed structural similarities between the N-terminal domain of RimP and the single domain of RbfA. RbfA is a 15 kDa protein that was found to high copy-suppress a dominant C23U 16S rRNA mutation giving rise to cold-sensitivity in E. coli. A number of chromosomal suppressor mutations that increased the growth rate of an rbfA null mutant were isolated. The five strongest suppressor mutations were localized to the rpsE gene, for r-protein S5 and resulted in amino acid substitutions in three positions: G87A, G87S, G91A, A127V and A127T. These alterations improved translation and the processing of 16S rRNA in the rbfA null mutant. Moreover, they also suppressed the slow growth of the C23U rRNA mutant at 30, 37 and 44°C.