Abstract
It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome.
Highlights
In the presence of oxygen largely remains to be elucidated[14,15]
We propose that a major functional role of ribosome-associated AdhE could be to promote local unwinding of some of the mRNAs with stable stem loop structure at the 3′ end, ensuring its linear configuration at the entrance (Fig. 5)
Fast kinetics of ribosome biogenesis and activity in vivo indicates that intra-cellular processes may be supplemented by additional factors capable of enhancing efficient assembly and protein synthesis[45,46]
Summary
In the presence of oxygen largely remains to be elucidated[14,15]. OmpC (~40 KDa), a cation-selective porin in E. coli, is known to form trans-membrane pores spanning the outer membrane[16,17]. Co-expression of the ompC gene with the gene of an rRNA modification enzyme has been identified in recent protein interaction network studies[4,6,21]. Our finding led us to propose that protein AdhE is likely involved in unwinding of specific, structured mRNAs at the 3′ end, at least under certain conditions. It was evidenced by Noller’s group that ribosomal proteins S3 and S4 possess weak mRNA helicase activity[22,23], so far, in bacteria, no ribosome-interacting factor has been identified that shows mRNA helicase activity upon binding at the mRNA entry path. We speculate that AdhE might exert its helicase activity in specific cellular environments
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