Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state

Zhou, Jie; Lancaster, Laura; Donohue, John Paul; Noller, Harry F.

doi:10.1073/pnas.1901310116

Title: Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state

Abstract

The elongation factor G (EF-G)–catalyzed translocation of mRNA and tRNA through the ribosome is essential for vacating the ribosomal A site for the next incoming aminoacyl-tRNA, while precisely maintaining the translational reading frame. Here, the 3.2-Å crystal structure of a ribosome translocation intermediate complex containing mRNA and two tRNAs, formed in the absence of EF-G or GTP, provides insight into the respective roles of EF-G and the ribosome in translocation. Unexpectedly, the head domain of the 30S subunit is rotated by 21°, creating a ribosomal conformation closely resembling the two-tRNA chimeric hybrid state that was previously observed only in the presence of bound EF-G. The two tRNAs have moved spontaneously from their A/A and P/P binding states into ap/P and pe/E states, in which their anticodon loops are bound between the 30S body domain and its rotated head domain, while their acceptor ends have moved fully into the 50S P and E sites, respectively. Remarkably, the A-site tRNA translocates fully into the classical P-site position. Furthermore, although the mRNA also undergoes movement, codon–anticodon interaction is disrupted in the absence of EF-G, resulting in slippage of the translational reading frame. We conclude that, although movement of both tRNAs and mRNA (alongmore »« less

Authors:

Zhou, Jie ^[1]; Lancaster, Laura ^[1]; Donohue, John Paul ^[1];

^[1]

Univ. of California, Santa Cruz, CA (United States)

Publication Date:: Mon Apr 01 00:00:00 EDT 2019

Research Org.:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Org.:: National Institutes of Health (NIH); Robert L. Sinsheimer Chair

OSTI Identifier:: 1578229

Grant/Contract Number:: R35-GM118156

Resource Type:: Accepted Manuscript

Journal Name:: Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:: Journal Volume: 116; Journal Issue: 16; Journal ID: ISSN 0027-8424

Publisher:: National Academy of Sciences

Country of Publication:: United States

Language:: ENGLISH

Subject:: 59 BASIC BIOLOGICAL SCIENCES; ribosomes; translocation; EF-G; frame shifting; translation

Citation Formats


                    Zhou, Jie, Lancaster, Laura, Donohue, John Paul, and Noller, Harry F. Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state.  United States: N. p., 2019. 
Web.  doi:10.1073/pnas.1901310116.

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                    Zhou, Jie, Lancaster, Laura, Donohue, John Paul, & Noller, Harry F. Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state.  United States.  https://doi.org/10.1073/pnas.1901310116

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                    Zhou, Jie, Lancaster, Laura, Donohue, John Paul, and Noller, Harry F. Mon .  
"Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state".  United States.  https://doi.org/10.1073/pnas.1901310116.  https://www.osti.gov/servlets/purl/1578229.

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@article{osti_1578229,

  title        = {Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state},

  author       = {Zhou, Jie and Lancaster, Laura and Donohue, John Paul and Noller, Harry F.},

  abstractNote = {The elongation factor G (EF-G)–catalyzed translocation of mRNA and tRNA through the ribosome is essential for vacating the ribosomal A site for the next incoming aminoacyl-tRNA, while precisely maintaining the translational reading frame. Here, the 3.2-Å crystal structure of a ribosome translocation intermediate complex containing mRNA and two tRNAs, formed in the absence of EF-G or GTP, provides insight into the respective roles of EF-G and the ribosome in translocation. Unexpectedly, the head domain of the 30S subunit is rotated by 21°, creating a ribosomal conformation closely resembling the two-tRNA chimeric hybrid state that was previously observed only in the presence of bound EF-G. The two tRNAs have moved spontaneously from their A/A and P/P binding states into ap/P and pe/E states, in which their anticodon loops are bound between the 30S body domain and its rotated head domain, while their acceptor ends have moved fully into the 50S P and E sites, respectively. Remarkably, the A-site tRNA translocates fully into the classical P-site position. Furthermore, although the mRNA also undergoes movement, codon–anticodon interaction is disrupted in the absence of EF-G, resulting in slippage of the translational reading frame. We conclude that, although movement of both tRNAs and mRNA (along with rotation of the 30S head domain) can occur in the absence of EF-G and GTP, EF-G is essential for enforcing coupled movement of the tRNAs and their mRNA codons to maintain the reading frame.},

  doi          = {10.1073/pnas.1901310116},

  journal      = {Proceedings of the National Academy of Sciences of the United States of America},

  number       = 16,

  volume       = 116,

  place        = {United States},

  year         = {Mon Apr 01 00:00:00 EDT 2019},

  month        = {Mon Apr 01 00:00:00 EDT 2019}

}

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Title: Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state

Abstract

Citation Formats

Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome journal, January 1997

Kinetics of Spontaneous and EF-G-Accelerated Rotation of Ribosomal Subunits journal, August 2016

Elongation Factor G Bound to the Ribosome in an Intermediate State of Translocation journal, June 2013

Structural aspects of messenger RNA reading frame maintenance by the ribosome journal, April 2010

Crystal Structure of a 70S Ribosome-tRNA Complex Reveals Functional Interactions and Rearrangements journal, September 2006

Coordinated conformational and compositional dynamics drive ribosome translocation journal, April 2013

Mechanism of tRNA-mediated +1 ribosomal frameshifting journal, September 2018

Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation journal, February 2016

Structure of EF-G–ribosome complex in a pretranslocation state journal, August 2013

Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation journal, December 2013

Role of Domains 4 and 5 in Elongation Factor G Functions on the Ribosome journal, July 2000

Molecular mechanics of 30S subunit head rotation journal, September 2014

Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding journal, May 2011

Translocation as continuous movement through the ribosome journal, November 2016

Elongation factor G initiates translocation through a power stroke journal, June 2016

Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome journal, October 2008

mRNA translocation occurs during the second step of ribosomal intersubunit rotation journal, March 2011

Recurring RNA structural motifs underlie the mechanics of L1 stalk movement journal, February 2017

Catalysis of Ribosomal Translocation by Sparsomycin journal, May 2003

Structures of modified eEF2·80S ribosome complexes reveal the role of GTP hydrolysis in translocation journal, April 2007

The ribosome moves: RNA mechanics and translocation journal, December 2017

How the ribosome hands the A-site tRNA to the P site during EF-G-catalyzed translocation journal, September 2014

Rotation of the head of the 30S ribosomal subunit during mRNA translocation journal, November 2012

Factor-free (“Non-enzymic”) and factor-dependent systems of translation of polyuridylic acid by Escherichia coli ribosomes journal, March 1976

Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy journal, July 2010

Structure of the ribosome with elongation factor G trapped in the pretranslocation state journal, December 2013

Conformational Changes of Elongation Factor G on the Ribosome during tRNA Translocation journal, January 2015

Messenger RNA interactions in the decoding center control the rate of translocation journal, October 2011

Spontaneous Intersubunit Rotation in Single Ribosomes journal, June 2008

Crystal Structures of EF-G–Ribosome Complexes Trapped in Intermediate States of Translocation journal, June 2013

EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding journal, January 2004

Choreography of molecular movements during ribosome progression along mRNA journal, March 2016

Movement of Elongation Factor G between Compact and Extended Conformations journal, January 2015

Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes journal, July 2008

Synchronous tRNA movements during translocation on the ribosome are orchestrated by elongation factor G and GTP hydrolysis: Insights & Perspectives journal, August 2014

EF-G catalyzes tRNA translocation by disrupting interactions between decoding center and codon–anticodon duplex journal, August 2014

tRNA dynamics on the ribosome during translation journal, August 2004

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome journal, July 2007

Intermediate states in the movement of transfer RNA in the ribosome journal, November 1989

The hybrid state of tRNA binding is an authentic translation elongation intermediate journal, February 2006

EFG-independent Translocation of the mRNA:tRNA Complex is Promoted by Modification of the Ribosome with Thiol-specific Reagents journal, December 2002

Following movement of domain IV of elongation factor G during ribosomal translocation journal, October 2014

Template-free ribosomal synthesis of polypeptides from aminoacyl-tRNA: Polyphenylalanine synthesis from phenylalanyl-tRNA journal, September 1986

Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy journal, October 2003

The Structure of the Ribosome with Elongation Factor G Trapped in the Posttranslocational State journal, October 2009

Crystal Structure of the Ribosome at 5.5 A Resolution journal, March 2001

GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits journal, March 2014

Structure of the 70S Ribosome Complexed with mRNA and tRNA journal, September 2006

A ratchet-like inter-subunit reorganization of the ribosome during translocation journal, July 2000

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites journal, December 2010

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation journal, December 2012

Conformational Changes of the Small Ribosomal Subunit During Elongation Factor G-dependent tRNA–mRNA Translocation journal, November 2004

Coordinated Conformational and Compositional Dynamics Drive Ribosome Translocation journal, January 2013

Thermodynamic control of −1 programmed ribosomal frameshifting journal, October 2019

Translational recoding: canonical translation mechanisms reinterpreted journal, September 2019

The energy landscape of −1 ribosomal frameshifting journal, January 2020

Active role of elongation factor G in maintaining the mRNA reading frame during translation journal, December 2019

Translational recoding: canonical translation mechanisms reinterpreted journal, September 2019

The energy landscape of −1 ribosomal frameshifting journal, January 2020

Active role of elongation factor G in maintaining the mRNA reading frame during translation journal, December 2019

The Impact of the Stringent Response on TRAFAC GTPases and Prokaryotic Ribosome Assembly journal, October 2019

Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome
journal, January 1997

Kinetics of Spontaneous and EF-G-Accelerated Rotation of Ribosomal Subunits
journal, August 2016

Elongation Factor G Bound to the Ribosome in an Intermediate State of Translocation
journal, June 2013

Structural aspects of messenger RNA reading frame maintenance by the ribosome
journal, April 2010

Crystal Structure of a 70S Ribosome-tRNA Complex Reveals Functional Interactions and Rearrangements
journal, September 2006

Coordinated conformational and compositional dynamics drive ribosome translocation
journal, April 2013

Mechanism of tRNA-mediated +1 ribosomal frameshifting
journal, September 2018

Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation
journal, February 2016

Structure of EF-G–ribosome complex in a pretranslocation state
journal, August 2013

Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation
journal, December 2013

Role of Domains 4 and 5 in Elongation Factor G Functions on the Ribosome
journal, July 2000

Molecular mechanics of 30S subunit head rotation
journal, September 2014

Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding
journal, May 2011

Translocation as continuous movement through the ribosome
journal, November 2016

Elongation factor G initiates translocation through a power stroke
journal, June 2016

Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome
journal, October 2008

mRNA translocation occurs during the second step of ribosomal intersubunit rotation
journal, March 2011

Recurring RNA structural motifs underlie the mechanics of L1 stalk movement
journal, February 2017

Catalysis of Ribosomal Translocation by Sparsomycin
journal, May 2003

Structures of modified eEF2·80S ribosome complexes reveal the role of GTP hydrolysis in translocation
journal, April 2007

The ribosome moves: RNA mechanics and translocation
journal, December 2017

How the ribosome hands the A-site tRNA to the P site during EF-G-catalyzed translocation
journal, September 2014

Rotation of the head of the 30S ribosomal subunit during mRNA translocation
journal, November 2012

Factor-free (“Non-enzymic”) and factor-dependent systems of translation of polyuridylic acid by Escherichia coli ribosomes
journal, March 1976

Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy
journal, July 2010

Structure of the ribosome with elongation factor G trapped in the pretranslocation state
journal, December 2013

Conformational Changes of Elongation Factor G on the Ribosome during tRNA Translocation
journal, January 2015

Messenger RNA interactions in the decoding center control the rate of translocation
journal, October 2011

Spontaneous Intersubunit Rotation in Single Ribosomes
journal, June 2008

Crystal Structures of EF-G–Ribosome Complexes Trapped in Intermediate States of Translocation
journal, June 2013

EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding
journal, January 2004

Choreography of molecular movements during ribosome progression along mRNA
journal, March 2016

Movement of Elongation Factor G between Compact and Extended Conformations
journal, January 2015

Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes
journal, July 2008

Synchronous tRNA movements during translocation on the ribosome are orchestrated by elongation factor G and GTP hydrolysis: Insights & Perspectives
journal, August 2014

EF-G catalyzes tRNA translocation by disrupting interactions between decoding center and codon–anticodon duplex
journal, August 2014

tRNA dynamics on the ribosome during translation
journal, August 2004

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome
journal, July 2007

Intermediate states in the movement of transfer RNA in the ribosome
journal, November 1989

The hybrid state of tRNA binding is an authentic translation elongation intermediate
journal, February 2006

EFG-independent Translocation of the mRNA:tRNA Complex is Promoted by Modification of the Ribosome with Thiol-specific Reagents
journal, December 2002

Following movement of domain IV of elongation factor G during ribosomal translocation
journal, October 2014

Template-free ribosomal synthesis of polypeptides from aminoacyl-tRNA: Polyphenylalanine synthesis from phenylalanyl-tRNA
journal, September 1986

Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy
journal, October 2003

The Structure of the Ribosome with Elongation Factor G Trapped in the Posttranslocational State
journal, October 2009

Crystal Structure of the Ribosome at 5.5 A Resolution
journal, March 2001

GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits
journal, March 2014

Structure of the 70S Ribosome Complexed with mRNA and tRNA
journal, September 2006

A ratchet-like inter-subunit reorganization of the ribosome during translocation
journal, July 2000

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites
journal, December 2010

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation
journal, December 2012

Conformational Changes of the Small Ribosomal Subunit During Elongation Factor G-dependent tRNA–mRNA Translocation
journal, November 2004

Coordinated Conformational and Compositional Dynamics Drive Ribosome Translocation
journal, January 2013

Thermodynamic control of −1 programmed ribosomal frameshifting
journal, October 2019

Translational recoding: canonical translation mechanisms reinterpreted
journal, September 2019

The energy landscape of −1 ribosomal frameshifting
journal, January 2020

Active role of elongation factor G in maintaining the mRNA reading frame during translation
journal, December 2019

Translational recoding: canonical translation mechanisms reinterpreted
journal, September 2019

The energy landscape of −1 ribosomal frameshifting
journal, January 2020

Active role of elongation factor G in maintaining the mRNA reading frame during translation
journal, December 2019

The Impact of the Stringent Response on TRAFAC GTPases and Prokaryotic Ribosome Assembly
journal, October 2019