Domain-specific recruitment of amide amino acids for protein synthesis.

The formation of aminoacyl-transfer RNA is a crucial step in ensuring the accuracy of protein synthesis. Despite the central importance of this process in all living organisms, it remains unknown how archaea and some bacteria synthesize Asn-tRNA and Gln-tRNA. These amide aminoacyl-tRNAs can be formed by the direct acylation of tRNA, catalysed by asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, respectively. A separate, indirect pathway involves the formation of mis-acylated Asp-tRNA(Asn) or Glu-tRNA(Gln), and the subsequent amidation of these amino acids while they are bound to tRNA, which is catalysed by amidotransferases. Here we show that all archaea possess an archaea-specific heterodimeric amidotransferase (encoded by gatD and gatE) for Gln-tRNA formation. However, Asn-tRNA synthesis in archaea is divergent: some archaea use asparaginyl-tRNA synthetase, whereas others use a heterotrimeric amidotransferase (encoded by the gatA, gatB and gatC genes). Because bacteria primarily use transamidation, and the eukaryal cytoplasm uses glutaminyl-tRNA synthetase, it appears that the three domains use different mechanisms for Gln-tRNA synthesis; as such, this is the only known step in protein synthesis where all three domains have diverged. Closer inspection of the two amidotransferases reveals that each of them recruited a metabolic enzyme to aid its function; this provides direct evidence for a relationship between amino-acid metabolism and protein biosynthesis.

A HU-like protein binds to specific sites within nod promoters of Rhizobium leguminosarum.

Nodulation genes (nod) of rhizobia are essential for establishment of its symbiosis with specific legume hosts and are usually located on the Sym(biosis) megaplasmid. In this work we identified a new Sym plasmid independent protein in Rhizobium leguminosarum, Px, by its ability to bind to nod promoters and induce DNA bending. Depending upon its concentrations relative to DNA templates, Px could either stimulate or inhibit in vitro transcription of the major regulatory nodulation gene nodD. This may result from its property to bind to specific sites within nod promoters at lower concentration or in the presence of competitor calf thymus DNA but nonspecifically associate with DNA at higher levels or in the absence of competitors. Its binding sites within nodD and nodF promoters were determined by DNase I footprinting but showed no sequence consensus. N-terminal sequencing and Western blot revealed that Px belongs to the HU class of prokaryotic histone-like proteins. Its binding feature and functioning mechanism were discussed in the light of this discovery.