期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 282, 期 48, 页码 34779-34786出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M707256200
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资金
- NIAID NIH HHS [P01 AI055621-050003, P01 AI055621, AI055621] Funding Source: Medline
- NIGMS NIH HHS [R01 GM065319-05, GM65319, R01 GM065319, R56 GM065319] Funding Source: Medline
Small protein B (SmpB) is a requisite component of the transfer messenger RNA (tmRNA)-mediated bacterial translational quality control system known as trans-translation. The initial binding of tmRNA and its subsequent accommodation into the ribosomal A-site are activities intimately linked to SmpB protein function. From a mechanistic perspective, two key unanswered questions that require further investigation are: 1) what constitutes a stalled ribosome recognition complex and 2) does SmpB pre-bind ribosomes to recruit tmRNA. We have assessed, both in vivo and in vitro, the nature and stability of free SmpB interactions with stalled ribosomes and examined whether these interactions are functionally relevant. We present evidence to demonstrate that interaction of free SmpB with ribosomes is salt sensitive and significantly more labile than interaction of the SmpB center dot tmRNA complex with ribosomes. Upon dissociation of 70 S ribosomes SmpB partitions primarily with tmRNA rather than ribosomal subunits. This finding is consistent with biochemical and structural data demonstrating that tmRNA is the high-affinity binding partner of SmpB. Moreover, we show that under normal physiological conditions roughly similar numbers of SmpB and tmRNA molecules are present in cells. Our investigations also reveal that upon induction of a nonstop mRNA, SmpB is enriched in stalled ribosome fractions only in the presence of tmRNA. Based on these findings, we conclude that SmpB does not pre-bind stalled ribosome and that functional SmpB-stalled ribosome interactions require tmRNA. We propose that a 1:1:1 complex of SmpB center dot tmRNA center dot EF-Tu(GTP) recognizes and binds a stalled ribosome to initiate trans-translation.
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