GGQ methylation enhances both speed and accuracy of stop codon recognition by bacterial class-I release factors

Autor: Shreya, Pundir, Xueliang, Ge, Suparna, Sanyal
Rok vydání: 2021
Předmět:
Models
Molecular
ATP
adenosine triphosphate
kcat
maximal rate of catalysis
BOP
BODIPYTM 576/589
Amino Acid Motifs
translation
RT-PCR
real-time polymerase chain reaction
Methylation
FRET
fluorescence resonance energy transfer
kcat/KM
catalytic efficiency
skin and connective tissue diseases
cryo-EM
cryo-electron microscopy
CD
circular dichroism
Conserved Sequence
GGQ motif
Tm
melting temperature
mRF
methylated release factor
accuracy
KM
Michaelis–Menten constant
Escherichia coli Proteins
release factor
Temperature
a.u.
arbitrary units
translation termination
EF
elongation factor
M-M
Michaelis–Menten
rRNA
ribosomal ribonucleic acid
MD
molecular dynamics
IF
initiation factor
rNTP
ribonucleoside triphosphate
Kinetics
ribosome
RF
release factor
Codon
Terminator
RC
release complex
Peptide Termination Factors
Research Article
Zdroj: The Journal of Biological Chemistry
ISSN: 1083-351X
Popis: 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.
Databáze: OpenAIRE
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