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Biomolecules 2017, 7(2), 39; doi:10.3390/biom7020039

TrmL and TusA Are Necessary for rpoS and MiaA Is Required for hfq Expression in Escherichia coli

1
Department of Microbiology, College of Medicine, Howard University, Washington, DC 20059, USA
2
Department of Biology, College of Arts and Sciences, Howard University, Washington, DC 20059, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Valérie de Crécy-Lagard
Received: 1 January 2017 / Revised: 31 March 2017 / Accepted: 12 April 2017 / Published: 4 May 2017
(This article belongs to the Special Issue tRNA Modifications: Synthesis, Function and Beyond)
View Full-Text   |   Download PDF [1505 KB, uploaded 4 May 2017]   |  

Abstract

Previous work demonstrated that efficient RNA Polymerase sigma S-subunit (RpoS) translation requires the N6-isopentenyladenosine i6A37 transfer RNA (tRNA) modification for UUX-Leu decoding. Here we investigate the effect of two additional tRNA modification systems on RpoS translation; the analysis was also extended to another High UUX-leucine codon (HULC) protein, Host Factor for phage Qβ (Hfq). One tRNA modification, the addition of the 2’-O-methylcytidine/uridine 34 (C/U34m) tRNA modification by tRNA (cytidine/uridine-2’O)-ribose methyltransferase L (TrmL), requires the presence of the N6-isopentenyladenosine 37 (i6A37) and therefore it seemed possible that the defect in RpoS translation in the absence of i6A37 prenyl transferase (MiaA) was in fact due to the inability to add the C/U34m modification to UUX-Leu tRNAs. The second modification, addition of 2-thiouridine (s2U), part of (mnm5s2U34), is dependent on tRNA 2-thiouridine synthesizing protein A (TusA), previously shown to affect RpoS levels. We compared expression of PBAD-rpoS990-lacZ translational fusions carrying wild-type UUX leucine codons with derivatives in which UUX codons were changed to CUX codons, in the presence and absence of TrmL or TusA. The absence of these proteins, and therefore presumably the modifications they catalyze, both abolished PBAD-rpoS990-lacZ translation activity. UUX-Leu to CUX-Leu codon mutations in rpoS suppressed the trmL requirement for PBAD-rpoS990-lacZ expression. Thus, it is likely that the C/U34m and s2U34 tRNA modifications are necessary for full rpoS translation. We also measured PBAD-hfq306-lacZ translational fusion activity in the absence of C/U34m (trmL) or i6A37 (miaA). The absence of i6A37 resulted in decreased PBAD-hfq306-lacZ expression, consistent with a role for i6A37 tRNA modification for hfq translation. View Full-Text
Keywords: s2U34 tRNA modification; C/U34m tRNA modification; i6A37 tRNA modification; RpoS; Hfq; MiaA; TrmL; TusA; leucine; codon bias s2U34 tRNA modification; C/U34m tRNA modification; i6A37 tRNA modification; RpoS; Hfq; MiaA; TrmL; TusA; leucine; codon bias
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Aubee, J.I.; Olu, M.; Thompson, K.M. TrmL and TusA Are Necessary for rpoS and MiaA Is Required for hfq Expression in Escherichia coli. Biomolecules 2017, 7, 39.

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