Ribosomes are the molecular machines that carry out protein biosynthesis in all living organisms. Bacterial ribosomes are composed of three different ribosomal RNAs (16S, 23S and 5S)and more than 50 different ribosomal proteins. The thermodynamics and kinetics of in vitro ribosomal assembly have been studied extensively. However, during ribosome biogenesis in vivo, ribosome assembly occurs concurrently with transcription, folding, post-transcriptional modifications and processing of rRNA. Unfortunately, the effects of nucleotide modifications and their respective modification enzymes on ribosomal assembly are understudied. My project in Abey lab is to investigate how RNA modification enzyme RsuA interacts with 16S rRNA and other primary assembly proteins such as S4, S17, and S20 during bacterial ribosome assembly. A fluorescence based assay was used to monitor the RsuA binding to the 16S rRNA in the presence of other ribosomal proteins that are binding near the RsuA binding site. We observed that protein RsuA is cooperative with protein S17 for its binding but anti-cooperative with protein S4. Furthermore, our results suggested that protein RsuA prefers extended h18 structure for its binding.
Ribosomes are the molecular machines that carry out protein biosynthesis in all living organisms. They are composed of three different ribosomal RNAs and more than 50 ribosomal proteins. My project in Abey lab is to investigate how RNA modification enzyme RsuA interacts with ribosomal proteins S4 and S17 in coordination with binding to 16S ribosomal RNA. To achieve this goal, I have developed an assay to monitor binding of RsuA to ribosomal RNA, which will allow us to determine the binding affinity of RsuA when other assembly proteins are present. This will then allow me to calculate the thermodynamic cooperativity between RsuA enzyme and ribosomal proteins. Our findings give us more insight to how modification enzymes modulate the hierarchy of protein addition during ribosome biogenesis.