Abstract
Accurate translation termination in bacteria requires correct recognition of the stop codons by the class-I release factors (RFs) RF1 and RF2, which release the nascent peptide from the peptidyl tRNA after undergoing a “compact to open” conformational transition. These RFs possess a conserved Gly-Gly-Gln (GGQ) peptide release motif, of which the Q residue is posttranslationally methylated. GGQ-methylated RFs have been shown to be faster in peptide release than the unmethylated ones, but it was unknown whether this modification had additional roles. Using a fluorescence-based real-time in vitro translation termination assay in a stopped-flow instrument, we demonstrate that methylated RF1 and RF2 are two- to four-fold more accurate in the cognate stop codon recognition than their unmethylated variants. Using pH titration, we show that the lack of GGQ methylation facilitates the “compact to open” transition, which results in compromised accuracy of the unmethylated RFs. Furthermore, thermal melting studies using circular dichroism and SYPRO-orange fluorescence demonstrate that GGQ methylation increases overall stability of the RF proteins. This increased stability, we suspect, is the basis for the more controlled conformational change of the methylated RFs upon codon recognition, which enhances both their speed and accuracy. This GGQ methylation-based modulation of the accuracy of RFs can be a tool for regulating translational termination in vivo.
Highlights
Termination is an important step of translation during which the nascent peptides are released from the ribosome
We used a ribosomal release complex (RC), which harbored in the P-site, a peptidyl tRNA carrying fluorescent BODIPY 576/589 (BOP) labeled Met-Leu-Leu tripeptide, and in the A-site, one of the three stop codons or the UGG codon specific for tryptophan (Trp), on the mRNA
When the same assay was performed with near-cognate RCUAG, release factor 2 (RF2) showed about twofold higher kcat than mRF2 with almost no change in Michaelis–Menten constant (KM) (Fig. 2I)
Summary
Termination is an important step of translation during which the nascent peptides are released from the ribosome. Molecular dynamics (MD) simulations-based studies [24] identified additional residues beyond the PXT and SPF motifs as crucial for correct stop codon recognition by RF1 and RF2. Among these the role of Arg213 of RF2 has been studied extensively by mutagenesis and fast kinetics [25]. Kinetics of the “compact to open” transition has been followed by state-of-the-art fluorescence resonance energy transfer (FRET) [36] and in silico simulations [9] These studies decipher a near complete mechanistic picture of translation termination by the bacterial class-I RFs, where the stop codon recognition and conformational change relate to the accuracy in termination, whereas the peptide release governs the speed of the process (Fig. 1C)
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