Small protein B interacts with the large and the small subunits of a stalled ribosome during trans-translation

During trans-translation, stalled bacterial ribosomes are rescued by small protein B (SmpB) and by transfer-messenger RNA (tmRNA). Stalled ribosomes switch translation from the defective messages to a short internal reading frame on tmRNA that tags the nascent peptide chain for degradation and recycles the ribosomes. We present evidences that SmpB binds the large and small ribosomal subunits in vivo and in vitro. The binding between SmpB and the ribosomal subunits is very tight, with a dissociation constant of 1.7 × 10(−10) M, similar to its K(D) for the 70S ribosome or for tmRNA. tmRNA displaces SmpB from its 50S binding but not from the 30S. In vivo, SmpB is detected on the 50S when trans-translation is impaired by lacking tmRNA or a functional SmpB. SmpB contacts the large subunit transiently and early during the trans-translational process. The affinity of SmpB for the two ribosomal subunits is modulated by tmRNA in the course of trans-translation. It is the first example of two copies of the same protein interacting with two different functional sites of the ribosomes

SmpB is detected on the 50S subunit when translation and -translation are blocked by chloramphenicol

<p><b>Copyright information:</b></p><p>Taken from "Small protein B interacts with the large and the small subunits of a stalled ribosome during -translation"</p><p>Nucleic Acids Research 2006;34(6):1935-1943.</p><p>Published online 12 Apr 2006</p><p>PMCID:PMC1435831.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Crude ribosomes from wild-type cells which were fractionated by sucrose gradient centrifugation at a low concentration of Mg ions. () tmRNA was detected by northern hybridization using complementary P-labeled DNA oligonucleotides. () The presence of endogenous SmpB was detected by western blotting using rabbit polyclonal antibody directed against a histidine-tagged SmpB