Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli

Abstract

Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA)⅐SmpB-mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pausing during termination. Streptomycin did not inhibit A-site cleavage in rpsL mutants, which express streptomycin-resistant variants of ribosomal protein S12. However, rpsL strains exhibited reduced A-site mRNA cleavage compared with rpsL ؉ cells. Additionally, tmRNA⅐SmpB-mediated SsrA peptide tagging was significantly reduced in several rpsL strains but could be fully restored in a subset of mutants when treated with streptomycin. The streptomycin-dependent rpsL(P90K) mutant also showed significantly lower levels of A-site cleavage and tmRNA⅐SmpB activity. Mutations in rpsD (encoding ribosomal protein S4), which suppressed streptomycin dependence, were able to partially restore A-site cleavage to rpsL(P90K) cells but failed to increase tmRNA⅐SmpB activity. Taken together, these results show that perturbations to A-site structure and function modulate A-site mRNA cleavage and tmRNA⅐SmpB activity. We propose that tmRNA⅐SmpB binds to streptomycinresistant rpsL ribosomes less efficiently, leading to a partial loss of ribosome rescue function in these mutants.

The tmRNA ribosome-rescue system

“…Holberger & Hayes examined the effects of streptomycin-resistant rpsL mutations on tmRNA activity (Holberger and Hayes, 2009). The rpsL gene encodes ribosomal protein S12, which is located near the decoding center and has long been known to influence translational fidelity (Kurland et al, 1996).…”

The role of SmpB and the ribosomal decoding center in licensing tmRNA entry into stalled ribosomes

“…Alternatively, the S12 protein is known to play an important role in the decoding process and may also influence tmRNA acceptance. S12 mutants can inhibit tmRNA tagging, although their mechanism of action is still unclear (Holberger and Hayes 2009;M Miller and A Buskirk, unpubl.). Experiments to determine the mechanisms by which SmpB activates the decoding machinery will likely yield more insight into transtranslation and perhaps canonical decoding as well.…”

Development and efficacy of a novel streptomycin-resistant Flavobacterium johnsoniae vaccine in grass carp (Ctenopharyngodon idella)

“…Mutations in the rpsL gene have been reported in streptomycin-resistant Gram-positive bacteria Mycobacterium smegmatis (Pelchovich et al, 2013) and Gram-negative bacteria Campylobacter jejuni (Olkkola et al, 2010). It was reported that an amino acid substitution (K42R, K42N, K42T, K42A, K42C, K42S, K42V, K42Y or P90F, P90Y, P90H, P90N) in the rpsL gene of Escherichia coli resulted in mRNA cleavage within the ribosomal A-site (Holberger and Hayes, 2009). The results of the present study suggest that R86W substitution in the rpsL gene was associated with streptomycin resistance.…”