PMID- 36282914
OWN - NLM
STAT- MEDLINE
DCOM- 20221027
LR  - 20230203
IS  - 1091-6490 (Electronic)
IS  - 0027-8424 (Print)
IS  - 0027-8424 (Linking)
VI  - 119
IP  - 44
DP  - 2022 Nov
TI  - The role of GTP hydrolysis by EF-G in ribosomal translocation.
PG  - e2212502119
LID - 10.1073/pnas.2212502119 [doi]
LID - e2212502119
AB  - Translocation of transfer RNA (tRNA) and messenger RNA (mRNA) through the 
      ribosome is catalyzed by the GTPase elongation factor G (EF-G) in bacteria. 
      Although guanosine-5'-triphosphate (GTP) hydrolysis accelerates translocation and 
      is required for dissociation of EF-G, its fundamental role remains unclear. Here, 
      we used ensemble Forster resonance energy transfer (FRET) to monitor how 
      inhibition of GTP hydrolysis impacts the structural dynamics of the ribosome. We 
      used FRET pairs S12-S19 and S11-S13, which unambiguously report on rotation of 
      the 30S head domain, and the S6-L9 pair, which measures intersubunit rotation. 
      Our results show that, in addition to slowing reverse intersubunit rotation, as 
      shown previously, blocking GTP hydrolysis slows forward head rotation. 
      Surprisingly, blocking GTP hydrolysis completely abolishes reverse head rotation. 
      We find that the S13-L33 FRET pair, which has been used in previous studies to 
      monitor head rotation, appears to report almost exclusively on intersubunit 
      rotation. Furthermore, we find that the signal from quenching of 3'-terminal 
      pyrene-labeled mRNA, which is used extensively to follow mRNA translocation, 
      correlates most closely with reverse intersubunit rotation. To account for our 
      finding that blocking GTP hydrolysis abolishes a rotational event that occurs 
      after the movements of mRNA and tRNAs are essentially complete, we propose that 
      the primary role of GTP hydrolysis is to create an irreversible step in a 
      mechanism that prevents release of EF-G until both the tRNAs and mRNA have moved 
      by one full codon, ensuring productive translocation and maintenance of the 
      translational reading frame.
FAU - Rexroad, Gillian
AU  - Rexroad G
AD  - Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 
      95064.
AD  - Department of Molecular, Cell and Developmental Biology, University of 
      California, Santa Cruz, CA 95064.
FAU - Donohue, John Paul
AU  - Donohue JP
AD  - Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 
      95064.
AD  - Department of Molecular, Cell and Developmental Biology, University of 
      California, Santa Cruz, CA 95064.
FAU - Lancaster, Laura
AU  - Lancaster L
AD  - Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 
      95064.
AD  - Department of Molecular, Cell and Developmental Biology, University of 
      California, Santa Cruz, CA 95064.
FAU - Noller, Harry F
AU  - Noller HF
AUID- ORCID: 0000-0002-2299-8700
AD  - Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 
      95064.
AD  - Department of Molecular, Cell and Developmental Biology, University of 
      California, Santa Cruz, CA 95064.
LA  - eng
GR  - R35 GM118156/GM/NIGMS NIH HHS/United States
GR  - R35-GM118156/HHS | NIH | National Institute of General Medical Sciences (NIGMS)/
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20221025
PL  - United States
TA  - Proc Natl Acad Sci U S A
JT  - Proceedings of the National Academy of Sciences of the United States of America
JID - 7505876
RN  - 0 (Peptide Elongation Factor G)
RN  - 86-01-1 (Guanosine Triphosphate)
RN  - 9014-25-9 (RNA, Transfer)
RN  - 0 (RNA, Messenger)
RN  - EC 3.6.1.- (GTP Phosphohydrolases)
RN  - 0 (Pyrenes)
RN  - 12133JR80S (Guanosine)
SB  - IM
MH  - *Peptide Elongation Factor G/genetics/chemistry
MH  - Guanosine Triphosphate/chemistry
MH  - Hydrolysis
MH  - *Ribosomes/metabolism
MH  - RNA, Transfer/chemistry
MH  - RNA, Messenger/chemistry
MH  - GTP Phosphohydrolases/genetics
MH  - Pyrenes/analysis
MH  - Guanosine
PMC - PMC9636962
OTO - NOTNLM
OT  - FRET
OT  - frameshifting
OT  - ribosome
OT  - translation
COIS- The authors declare no competing interest.
EDAT- 2022/10/26 06:00
MHDA- 2022/10/28 06:00
PMCR- 2022/10/25
CRDT- 2022/10/25 14:18
PHST- 2022/10/25 14:18 [entrez]
PHST- 2022/10/26 06:00 [pubmed]
PHST- 2022/10/28 06:00 [medline]
PHST- 2022/10/25 00:00 [pmc-release]
AID - 202212502 [pii]
AID - 10.1073/pnas.2212502119 [doi]
PST - ppublish
SO  - Proc Natl Acad Sci U S A. 2022 Nov;119(44):e2212502119. doi: 
      10.1073/pnas.2212502119. Epub 2022 Oct 25.