подсекция «цикл наук о живом

Ломоносов-2008 «Химия» Цикл наук о живом
One SmpB molecule accompanies tmRNA during its passage through the stalled
ribosomes
Bugaeva Elizaveta Yu. 1 , Shpanchenko Olga V. 2 , Dontsova Olga A. 2
1 Department of Bioinformatics and Bioengineering, M.V. Lomonosov Moscow State
University, 119992, Moscow, Russia.
2 Department of Chemistry, M.V. Lomonosov Moscow State University, 119992, Moscow,
Russia.
beu@rambler.ru
Trans-translation is an important protein synthesis quality control mechanism in bacteria
(reviewed by Moore and Sauer, 2007). It recycles ribosomes arrested during the translation of
non-stop mRNAs and directs the problematic mRNA and corresponding protein product for
degradation. Along with the common participants of the translation two additional players,
namely tmRNA (transfer-messenger RNA, or 10Sa RNA, or SsrA RNA) and SmpB (small
protein B), are required for trans-translation. It has been shown that two SmpB molecules bind
the ribosome during the initiation of trans-translation (Hallier et al., 2006; Kaur et al., 2006).
However, an alternative study suggests that only one molecule of SmpB, in complex with
tmRNA, interacts with the ribosome (Sundermeier et al., 2007).
Earlier we have developed a system to block tmRNA-ribosome complexes in vivo during
different stages of trans-translation (Shpanchenko et al., 2005). The stalled complexes were
isolated using affinity chromatography and the ratio of tmRNA, SmpB and ribosomes has been
determined using PAGE and blotting techniques.
To address the stoichiometry of SmpB during the process of trans-translation, we have
used this system to prepare a number of tmRNA-ribosomal complexes: blocked after the first
translocation step of messenger RNA-like domain (MLD) of tmRNA (stop codon at the 2 nd
position, leading to a two aminoacid-tag), two complexes stopped at the middle part of MLD
(4 th and 5 th codons, leading to a 4 and 5 aminoacid-tag, respectively) and one at the very end of
MLD (11 th codon). Using these complexes, we could more precisely determinate the
stoichiometry of SmpB within the stalled tmRNA-ribosome complexes, with the help of
Western blot analysis using antibodies raised against SmpB and the ribosomal protein S3, as
internal control.
The results suggest that only one molecule of SmpB remains bound to tmRNA and
accompanies it on its passage through the ribosome during the process of trans-translation.
References:
1 Hallier M., Desreac J., Felden B. 2006. Small protein B interacts with the large and the
small subunits of a stalled ribosome during trans-translation. NAR, 34: 1935-1943.
2 Kaur S., Gillet R., Li W., Gursky R., Frank J. 2006. Cryo-EM visualization of transfer
messenger RNA with two SmpB in the stalled ribosome. PNAS, 103: 16484-16489.
3 Moore, S. D., and Sauer, R. T. (2007). The tmRNA System for Translational
Surveillance and Ribosome Rescue. Annu Rev Biochem. 76:101-24.
4 Shpanchenko O. V., Zvereva M. I., Ivanov P. V., Bugaeva E. Yu., Rozov A. S.,
Bogdanov A. A., Kalkum M., Isaksson L., Nierhaus K. H., Dontsova O. A. 2005. Stepping
tmRNA through the ribosome. J.Biol.Chem., 280: 18368-18374.
5 Sundermeier T.R., Karzai A.W. 2007. Functional SmpB-ribosome interaction requires
tmRNA. JBC, 282: 34779-34786.
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