Characterization of the role of RbfA and KsgA in ribosome function

Sammanfattning: Ribosome biogenesis is the maturation and accurate assembly of ribosomal RNA and proteins to form functional ribosomes competent to enter the translation cycle, facilitated by the action of ribosome biogenesis factors. RbfA and KsgA are two factors involved in late stages of the 30S subunit biogenesis, yet before translation initiation. Despite the extensive research on both factors, their precise mechanisms remain unclear in translation initiation. Initiation factors constitute a key component of the translation initiation system, especially IF3 that proofreads the codon-anticodon interaction of the mRNA and initiator tRNA to maintain the accuracy of the initiation step. However, defective ribosomes with knockout RbfA (ΔrbfA) or KsgA (ΔksgA) appear to weaken IF3 binding affinities and subsequent proofreading. Previous results suggest that when defective ribosomes enter in vivo translation with cognate (AUG) or even near-cognate (AGG) start codons could bypass the fidelity checkpoints created by initiation factors, leading to the inaccurate initiation of translation. This study aimed to characterize the assembly defective ΔrbfA and ΔksgA ribosomes in in vitro initiation assays on cognate and near-cognate codons and with the interplay of initiation factors for the rate and accuracy in translation initiation. The two assays, BOP-Met-tRNAfMet binding and subunit association, reveal that ΔrbfA ribosomes are defective in both rate and accuracy in translation initiation, which largely contribute to its growth defect at 37°C. In contrast, ΔksgA ribosomes are not defective in the initiation step of translation when comparing to the wildtype ribosome. Therefore, RbfA seems to have the primary role in ribosome biogenesis compared to KsgA. Further, the results show that the initiation factors, in particular IF3, adds to the fidelity of initiation by dissociating the initiator tRNA on near/non-cognate codons, with no major difference between Wt and mutant ribosomes in selection against near/non-cognate codons. Therefore, we do not see any loss of fidelity for the ΔrbfA and ΔksgA ribosomes in in vitro initiation based assays, contrary to previous in vivo based results.