Thesis (Ph. D.)--University of Rochester. Department of Biology, 2015.
Ribonucleoproteins (RNPs) are present in all branches of life and perform vital functions required for cell viability. One such RNP is the bacterial ribosome. Composed of three rRNA and over 50 r-proteins this macromolecular complex must be assembled accurately and efficiently for proper translation and
subsequently cell viability. Fascinatingly, the Small SubUnit (SSU) of E. coli can be reconstituted in vitro from the core components of 16S rRNA and 20 r-proteins. This has given vital information on the form and function of the SSU, but the majority is from E. coli. To expand the current knowledge of SSU assembly, we have studied in vitro SSU assembly of two thermophilic bacteria, G. kaustophilus and T. thermophilus in two different ways. We first looked to compare the temperature dependent assembly of SSUs from E. coli, G. kaustophilus and T. thermophilus. We found, in all three organisms, that at least
two, distinct temperature dependent intermediates formed. These intermediate RNPs are incapable of proper translation indicating that there may be a conserved pathway within the 16S rRNA and TP30 components which help to ensure that improperly assembled SSUs remain unable to enter the translation
cycle.
To further our understanding of the rRNA and r-protein interactions within SSUs from E. coli and the thermophilic bacteria G. kaustophilus and T. thermophilus, we used a system of hybrid reconstitution. This study reconstituted 16S rRNA from one organism and r-proteins from another to form SSUs. We show that the temperature dependent assembly of hybrid SSUS is dependent on the optimum growth and reconstitution temperature of organism from which the r-proteins are purified. This indicates the proper folding and assembly of at least one r-protein drives the temperature dependence of assembly.
These studies do a great deal to expand on the current knowledge on SSU assembly in a diverse range of bacteria as well our understanding of formation of RNPs in general.
