Skip to main content
Figure 6 | BMC Molecular Biology

Figure 6

From: Requirements for resuming translation in chimeric transfer-messenger RNAs of Escherichia coli and Mycobacterium tuberculosis

Figure 6

In vivo tagging of truncated ribosomal protein L27 by hybrid tmRNAs in the presence of E. coli SmpB protein. (A) Lysates of cells expressing E. coli (Ec), M. tuberculosis (Mt) and hybrid tmRNAs (E1-E9) were fractionated on a 12.5% SDS polyacrylamide gel. Truncated and tagged proteins were detected by staining the gel with Coomassie Blue. L27 and L27* denote truncated and tagged ribosomal protein L27, respectively. Molecular markers of 6.0, 14.4 and 21.5 kDa are shown on the left side of the gel image. (B) Western analysis of the ribosome-bound E. coli SmpB. Aliquots of 1.0 A260 units of 70S ribosomes derived from lysates of IPTG-induced E. coli IW764 cells harboring plasmid pWOW derivatives were fractionated on a 10% SDS-polyacrylamide gel. The SmpB protein was visualized by Western blotting with polyclonal anti-E. coli SmpB antibodies. (C) Northern analysis of the ribosome-bound tmRNAs present in the tagging reaction mixture. Aliquots of 0.5 μg RNA extracted from 70S ribosomes were separated on a 6% denaturing polyacrylamide gel and blotted to a Zeta-probe membrane. [5’-32P]-labeled oligonucleotide probes complementary to a segment of M. tuberculosis tmRNA were hybridized to each tmRNA. To estimate quantity of tmRNA in the tmRNA:ribosome complexes, increasing amounts of purified M. tuberculosis tmRNA(H6) (0.0125, 0.025, 0.05, 0.1 and 0.2 pmoles) were also fractionated.

Back to article page