Characterizing an engineered release factor capable of reading all three stop codons (569.2)

Abstract

Translation termination is the last step of protein synthesis where the newly synthesized protein is released from the ribosome. For this, a class I release factor (RF1 and RF2) in bacteria binds to one of the three stop codons (UAA, UAG and UGA) and hydrolyze the ester bond between nascent protein and the peptidyl‐tRNA. RF1 and RF2, although similar to each other, read the stop codons in a semi specific manner i.e. RF1 reads UAA and UAG, and RF2 reads UAA and UGA codons. According to current literature [1] these factors discriminate highly against sense codons. Although molecular dynamics simulation experiments [2] have proposed a network of interactions through which RF1 and RF2 recognizes their respective stop codons, there exist very little biochemical experiments to explain how this high specificity is achieved. Using site directed mutagenesis we generated several variants of RF1 and RF2 and characterized those in competition and kinetic assays. One of our engineered release factors showed omnipotent property in stop codon recognition. We have characterized this engineered release factor with respect to other related codons especially in comparison with other release factors. Further we were able to validate our biochemical experiments using computer simulations. Our experiments pinpointed the crucial residues on the class‐I release factors for stop codon specificity and enabled us to engineer a class I release factor capable of reading all three stop codons. Grant Funding Source: Supported by; Swedish Research Council, Sven and Lilly Lawski Foundation

Structure-Based Energetics of Stop Codon Recognition by Eukaryotic Release Factor

“…Premature termination leads to disease. , The high accuracy of translation termination by release factor could be achieved from two aspects: (a) a binding affinity difference between cognate and near-cognate codons on the ribosome, K M and (b) facile eRF1-catalyzed hydrolysis of polypeptide chains from P-site tRNAs for the cognate codon, K cat . However, binding has been reported to be more crucial for bacteria. , In bacteria, two release factors RF1 (recognizes UAA and UAG) and RF2 (recognizes UAA and UGA) ensure discrimination against the sense codons. − Medium resolution bacterial termination complexes (3–3.5 Å) , had shown extensive protein–RNA interactions and provided the basis for computational analysis leading to quantification of the discriminatory power of the release factor for stop codons against sense codons. − Unlike sense codons, the third base of the bacterial stop codon stack with the ribosomal G530 of 16S rRNA instead of stacking with the first two bases.…”