Ribosomes accounts for approximately 40% of cellular dry mass. Biosynthesis of competent ribosomes is vital for cell survival. Scrupulous coordination among various processes such as transcription of ribosomal RNA, RNA folding, RNA processing and editing, and assembly of ribosomal proteins are required to make functional ribosomes. In Bacteria, protein assembly factors including RNA helicases, ATPases, RNA chaperones, and nucleotide modification enzymes work to synchronize these various biological processes occurring during ribosome biogenesis. Interestingly, several assembly factors such as RbfA do not possess enzymatic activity, whereas several other assembly factors perform important functions in the ribosome biogenesis other than their enzymatic activity. For example, the methyltransferase activity of KsgA enzyme is dispensable, whereas its quality control activity during ribosome biogenesis is critical for cell survival. Our research is focused on understanding important roles played by RNA modification enzymes such as RsmC, RsmG, and RsuA during ribosome biogenesis. Furthermore, we investigate how RNA modification enzymes perturb the energy landscape for 30S ribosome assembly and how rRNA nucleotide modifications modulate RNA-protein interactions using various biochemical and biophysical methods.
Post-transcriptional RNA modifications, ribosome biogenesis, and streptomycin resistance illustrate a strong correlation. It is known that mutations in rRNA modification enzymes influence antibiotic susceptibility and virulence in pathogenic bacteria. The study of post-transcriptional modifications is important as they are linked to many rare diseases in humans. Lack of pseudouridylation in human telomere RNA causes a rare congenital disease, X-linked dyskaryosis congenita. Recent studies illustrate elevated pseudouridine levels in yeast mRNA under starvation and heat shock conditions and in cancer cells. Furthermore, m6A modifications in mRNA are related to many human diseases and disorders. These results lead to the understanding that post-transcriptional modifications introduce a layer of regulation in eukaryotes.
Our preliminary studies indicate that protein RsmC can also function as an RNA chaperone protein thus increase the annealing rate of the two 16S helix 34 rRNA strands. We are extending our study to understand how RsmC enzyme chaperone activity is synchronized with rRNA transcription and the assembly of ribosomal proteins that bind to 30S 3’-major (head) domain. For this research, we will develop in vitro co-transcriptional assays that will mimic ribosome biogenesis in cells. These experiments will also indicate the existence of thermodynamic and kinetic cooperativity of binding between protein RsmC and various other ribosomal proteins and assembly factors including RNA modification enzymes. Secondly, we are exploring the possibility of inhibiting bacterial ribosome biogenesis by targeting modification enzymes. We will screen various peptide and phage display libraries to discover small peptides that can inhibit the ability of protein RsmC to bind to its target RNA. Our long-term goal is to rationally design peptide-based small molecular drugs to inhibit the functions of RNA methyltransferase enzymes that related various human diseases and disorders.
This project is aimed at understanding the molecular mechanism of how the nucleotide modification enzymes and/or their respective nucleotide modifications influence streptomycin resistance in bacteria.