No abstract

The gene coding for the protein methyl transferase HemK (ECBD_2409, 834 bp, 277 aa) was amplified from the genomic DNA of E. coli BL21 DE3 by colony PCR using the primers 5'-GTCCGAGCAGGACATATGGAATATCAA-3' and 5'-GCAGTGTAG AAAAACCTCGAGTTGATAAT-3', digested with NdeI and XhoI (New England Biolabs) and ligated into a pET-24a Vector (Novagen). The gene sequence encoding for the N-terminal domain of E. coli HemK (residues 1-73, HemK NTD hereafter) was amplified by PCR from a vector encoding full-length HemK (residues 1-277), using primers containing recognition sequences for NdeI and XhoI restriction enzymes for subsequent cloning into expression vector pET24-a (Novagen). The purified PCR fragment was digested with NdeI and XhoI restriction enzymes for 1 h and ligated into the expression vector pET-24a linearized with the same restriction enzymes. The Leu-Glu sequence (encoded by the XhoI restriction used for cloning) was subsequently removed by inverse deletion PCR. Mutants of wild type NTDHemK were made by PCR using a commercial site-directed mutagenesis kit (Stratagene, Carlsbad, CA) according to the manufacturer's instructions. We note that HemK from E. coli BL21 DE3 contains Lys at position 34, in contrast to HemK from E. coli K12, which contains Arg at position 34 and the structure of which is available. In the following, the sequence ofHemK from E. coli BL21 DE3 is denoted as wild type (wt). Endogenous tryptophan residues at position 6 and 78 were replaced with phenylalanine in all constructs using the quick-change method. For PET experiments tryptophan residues were introduced at position 6, 38, and 49 using site-directed mutagenesis. All constructs were verified by sequencing. Expression and purification of HemK NTDThe plasmid encoding HemK NTD (or its mutants) was transformed into competent E. coli Fluorescence emission spectra and equilibrium chemical unfoldingFluorescence emission spectra were recorded at 25 °C with a Horiba model Fluorolog 3 spectrofluorimeter connected to a circulating water bath. Measurements were performed in a quartz cuvette of 1-cm path length at a protein concentration of 2 µM in buffer E.Fluorescence was excited at 280 nm. Fluorescence emission spectra were collected between 310 and 420 nm in 1 nm increments and were corrected for background (buffer without protein). Slid widths of 1 and 10 nm were used for excitation and emission, respectively. Samples were incubated for 2 hours before measurements were taken. For each collected background-corrected fluorescence emission spectrum, the normalized global emission

Elongation factor G (EF-G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF-G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF-G mutants and translocation-specific antibiotics to investigate timing and energetics of translocation. We show that EF-G-GTP facilitates synchronous movements of peptidyl-tRNA on the two subunits into an early post-translocation state, which resembles a chimeric state identified by structural studies. EF-G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF-G. Our results reveal two distinct modes for utilizing the energy of EF-G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit.

No abstract

Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor.