Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release

Abstract

SignificanceTranslation termination is a crucial process during protein synthesis. Class I release factors (RFs) are in charge of recognizing stop codons and consequently hydrolyzing the peptidyl-tRNA at the ribosomal P site. High-resolution crystal- and cryo-EM structures of RFs bound to the ribosome revealed a network of potential interactions that is formed between the mRNA and RFs; however, it remained enigmatic which interactions are critical for accurate stop codon recognition and peptide release. By using chemically modified stop codon nucleotides, the importance and the contribution of single hydrogen bonds to stop codon recognition was investigated. This approach revealed a detailed picture of chemical groups defining a stop codon and contributing to the discrimination against sense codons during prokaryotic and eukaryotic translation termination.

Translation of non-standard codon nucleotides reveals minimal requirements for codon-anticodon interactions

“…This was achieved by employing chemically synthesized oligonucleotides harboring the desired base modification. Due to the length limitations of these oligonucleotides, a splinted ligation was carried out covalently linking the modified 3′-fragment to a 5′-transcript, resulting in a full-length mRNA 32 , 33 . The ligation products were purified and subsequently served as templates for translation.…”

“…The ligation products were purified and subsequently served as templates for translation. To investigate bacterial and eukaryotic translation processes, the recombinant PURExpress system (NEB) 34 and HEK293T cells were employed, respectively 32 , 33 .…”

“…Purine, 2,6-diaminopurine, 2-aminopurine, inosine, and ribose-abasic-modified oligonucleotides were purchased from Dharmacon. Zebularine and 2-pyridone-modified oligonucleotides were synthesized in-house 33 . Unmodified oligonucleotides were purchased from IDT.…”

“…Transcripts without a 5′-cap were synthesized employing the HiScribe™ T7 High Yield RNA synthesis kit (NEB, E2040S) as described by the manufacturer. The oligonucleotides used for assaying translation in HEK293T cells were poly(A)-tailed using the A-Plus™ Poly(A) Polymerase tailing kit (CELLSCRIPT, C-PAP5104H) 33 .…”

A molecular‐level perspective on the frequency, distribution, and consequences of messenger RNA modifications

“…Initial in vitro studies of varying resolution on a limited set of mRNA modifications indicate that modifications can alter the overall rate and fidelity of protein synthesis (Choi et al, , ; Eyler et al, ; Hoernes, Clementi, et al, ; Hoernes et al, , ; Hudson & Zaher, ; You et al, ). In fully reconstituted Escherichia coli translation systems, naturally occurring enzymatic nucleoside modifications and damaged ribonucleobases change translation to varying degrees (Figure ).…”