Abstract
Formation of the initiation translation complex containing the three initiation factors, IF1, IF2, and IF3, tRNA(fMet), and GTP constitutes the earliest event in the protein synthesis. IF2, a GTP-binding protein, is the principal factor involved in selecting and binding fMet-tRNA(fMet) to the 30 S ribosomal subunit. Although some chloroplast initiation translational factors have been identified and purified from algae, none of these factors have been characterized from plants. In this work, we report the molecular characterization of a nuclear-encoded chloroplastic IF2 gene from common bean (PvIF2cp). We show that the PvIF2cp gene encodes a protein containing a chloroplast translocation signal peptide, able to target a green fluorescent protein fusion protein to chloroplasts. A high accumulation of PvIF2cp transcript was found in photosynthetic tissues, whereas low mRNA levels were detected in etiolated plants and in nonphotosynthetic organs. Additional data indicate that the PvIF2cp transcript accumulation is modulated by light. The PvIF2cp gene encodes a functional factor, since the PvIF2cp conserved region, containing the G-domain and the C-terminal end, complements an Escherichia coli infB null mutation. Phylogenetic analysis using the PvIF2cp conserved region suggests that the PvIF2cp gene originated via endosymbiotic gene transfer to the nucleus and that it may be a useful marker for phylogeny reconstruction.
Highlights
The synthesis of prokaryotic proteins requires complex interactions between the different translation factors, ribosomal subunits, nucleotides, and tRNAs
Molecular Cloning of a Gene Encoding a Eubacterial-like Translational Initiation Factor-2 from Ph. vulgaris—By screening a bean water-stressed leaf cDNA library, several cDNA clones down-regulated by water deficit were isolated [27]
According to the endosymbiosis theory, the chloroplast lost most of its self-encoded functions to the nuclear genome during its evolution
Summary
Plant Material and Growth Conditions—Phaseolus vulgaris L. cv. negro Jamapa seeds were kindly provided by Dr J. In Vivo Complementation of an E. coli infB Null Mutation—An N terminus deletion of the PvIF2cp gene was constructed by polymerase chain reaction using the PvIF2cp cDNA as template and specific primers designed to incorporate an additional NcoI site. This modification did not generate any change in the amino acid sequence and left the methionine at position 411 as the initial amino acid. In vivo complementation experiments were carried out as described [34] This protocol is based on the presence of a thermosensitive lysogen containing the infB gene in the SL598R strain, which lacks a functional chromosomal IF2.
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