Nucleic Acids Research Advance Access published online on October 24, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl752
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Further insights into the tRNA modification process controlled by proteins MnmE and GidA of Escherichia coli
1 Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe Avda. Autopista del Saler 16-3, 46013 Valencia, Spain 2 Department of Molecular Biology, Umeå University S90187 Umeå, Sweden
*To whom correspondence should be addressed. Tel: +34 963289680; Fax: +34 963289701; Email: armengod{at}cipf.es
Received April 21, 2006. Revised August 3, 2006. Accepted September 23, 2006.
In Escherichia coli, proteins GidA and MnmE are involved in the addition of the carboxymethylaminomethyl (cmnm) group onto uridine 34 (U34) of tRNAs decoding two-family box triplets. However, their precise role in the modification reaction remains undetermined. Here, we show that GidA is an FAD-binding protein and that mutagenesis of the N-terminal dinucleotide-binding motif of GidA, impairs capability of this protein to bind FAD and modify tRNA, resulting in defective cell growth. Thus, GidA may catalyse an FAD-dependent reaction that is required for production of cmnmU34. We also show that GidA and MnmE have identical cell location and that both proteins physically interact. Gel filtration and native PAGE experiments indicate that GidA, like MnmE, dimerizes and that GidA and MnmE directly assemble in an 
2ß2 heterotetrameric complex. Interestingly, high-performance liquid chromatography (HPLC) analysis shows that identical levels of the same undermodified form of U34 are present in tRNA hydrolysates from loss-of-function gidA and mnmE mutants. Moreover, these mutants exhibit similar phenotypic traits. Altogether, these results do not support previous proposals that activity of MnmE precedes that of GidA; rather, our data suggest that MnmE and GidA form a functional complex in which both proteins are interdependent.
Present address: Lucía Yim, Instituto de Higiene, Facultad de Medicina, Universidad de la República, Avda. Alfredo Navarro 3051, 11600 Montevideo, Uruguay
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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