Frameshifting Stimulatory Sequence Induces Large Structural Change of Ribosomal Proteins When Bound to E. coli Ribosomes

Emily Armbruster; Kevin L. Weiss; Loukas Petridis; Jose Borreguero Calvo; James Byrnes; Alexis N. Williams; Peter Cornish; Sai Venkatesh Pingali

doi:10.1021/acs.biomac.4c01829

Frameshifting Stimulatory Sequence Induces Large Structural Change of Ribosomal Proteins When Bound to E. coli Ribosomes

Emily Armbruster
, Kevin L. Weiss
, Loukas Petridis
, Jose Borreguero Calvo
, James Byrnes
, Alexis N. Williams
, Peter Cornish
, Sai Venkatesh Pingali

Research output: Contribution to journal › Article › peer-review

Abstract

Biological macromolecular machines occupy a continuum of structural conformations to perform cellular tasks. Mapping this conformational space provides an insight into its functionality. While the cryo-electron microscopy resolution revolution has expanded our ability to characterize the conformational continuums, there are obstacles in structurally characterizing regions of high flexibility. These technical barriers have impeded characterization of flexible ribosomal proteins when the ribosome is interacting with mRNA stem-loop structures such as a frameshifting stimulatory sequence (FSS). Small-angle neutron/X-ray scattering and electron microscopy were used to study ribosomal samples and compared structural differences between a ribosome that is bound to an FSS stem-loop compared to a ribosome bound to linear mRNA. This comparison shows that a large protein stalk elongates by 22% when the 70S interacts with an mRNA stem-loop. Our results suggest that ribosomal proteins have extensive flexibility and may influence important ribosomal mechanisms, such as those that involve FSS.

Original language	English
Pages (from-to)	5571-5580
Number of pages	10
Journal	Biomacromolecules
Volume	26
Issue number	9
DOIs	https://doi.org/10.1021/acs.biomac.4c01829
State	Published - Sep 8 2025

Funding

E.A. was funded to spend 2 years of her doctoral work at Oak Ridge National Lab to prepare ribosomal samples and perform neutron scattering experiments through the Neutron Sciences Division Go! Student program, UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. E.A. and P.C. acknowledge the support offered from Helmut Kaiser and Haskell Taub and the National Science Foundation’s interdisciplinary IGERT Program “Neutron Scattering for the Science and Engineering of the 21st Century” and NSF MCA award MCB-2122902. This research used the LiX beamline (16-ID) of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE by Brookhaven National Laboratory under Contract No. DE-SC0012704. The LiX beamline is part of the Center for BioMolecular Structure (CBMS), which is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a Center Core Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1607011). LiX also received additional support from NIH Grant S10 OD012331. Neutron scattering research conducted at the Bio-SANS CG-3 instrument (Center for Structural Molecular Biology), a DOE Office of Science, Office of Biological and Environmental Research resource (FWP ERKP291), used resources at the High Flux Isotope Reactor, a DOE Office of Science, and EQ-SANS BL-6 instrument at the Spallation Neutron Source, Scientific User Facility operated by the Oak Ridge National Laboratory. This research was, in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. Negative stain reconstruction was conducted at the Center for Nanophase Materials Sciences, which is a US Department of Energy Office of Science User Facility at Oak Ridge National Laboratory. This manuscript has been coauthored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). FWP ERKP291, The Center for Structural Molecular Biology, DOE Office of Science, Office of Biological and Environmental Research resource. Go! Student program, Neutron Scattering Division, Oak Ridge National Laboratory E.A. was funded to spend 2 years of her doctoral work at Oak Ridge National Lab to prepare ribosomal samples and perform neutron scattering experiments through the Neutron Sciences Division Go! Student program, UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. E.A. and P.C. acknowledge the support offered from Helmut Kaiser and Haskell Taub and the National Science Foundation’s interdisciplinary IGERT Program “Neutron Scattering for the Science and Engineering of the 21st Century” and NSF MCA award MCB-2122902. This research used the LiX beamline (16-ID) of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE by Brookhaven National Laboratory under Contract No. DE-SC0012704. The LiX beamline is part of the Center for BioMolecular Structure (CBMS), which is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a Center Core Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1607011). LiX also received additional support from NIH Grant S10 OD012331. Neutron scattering research conducted at the Bio-SANS CG-3 instrument (Center for Structural Molecular Biology), a DOE Office of Science, Office of Biological and Environmental Research resource (FWP ERKP291), used resources at the High Flux Isotope Reactor, a DOE Office of Science, and EQ-SANS BL-6 instrument at the Spallation Neutron Source, Scientific User Facility operated by the Oak Ridge National Laboratory. This research was, in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. Negative stain reconstruction was conducted at the Center for Nanophase Materials Sciences, which is a US Department of Energy Office of Science User Facility at Oak Ridge National Laboratory. This manuscript has been coauthored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ).

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title = "Frameshifting Stimulatory Sequence Induces Large Structural Change of Ribosomal Proteins When Bound to E. coli Ribosomes",

abstract = "Biological macromolecular machines occupy a continuum of structural conformations to perform cellular tasks. Mapping this conformational space provides an insight into its functionality. While the cryo-electron microscopy resolution revolution has expanded our ability to characterize the conformational continuums, there are obstacles in structurally characterizing regions of high flexibility. These technical barriers have impeded characterization of flexible ribosomal proteins when the ribosome is interacting with mRNA stem-loop structures such as a frameshifting stimulatory sequence (FSS). Small-angle neutron/X-ray scattering and electron microscopy were used to study ribosomal samples and compared structural differences between a ribosome that is bound to an FSS stem-loop compared to a ribosome bound to linear mRNA. This comparison shows that a large protein stalk elongates by 22\% when the 70S interacts with an mRNA stem-loop. Our results suggest that ribosomal proteins have extensive flexibility and may influence important ribosomal mechanisms, such as those that involve FSS.",

author = "Emily Armbruster and Weiss, \{Kevin L.\} and Loukas Petridis and \{Borreguero Calvo\}, Jose and James Byrnes and Williams, \{Alexis N.\} and Peter Cornish and Pingali, \{Sai Venkatesh\}",

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T1 - Frameshifting Stimulatory Sequence Induces Large Structural Change of Ribosomal Proteins When Bound to E. coli Ribosomes

AU - Armbruster, Emily

AU - Weiss, Kevin L.

AU - Petridis, Loukas

AU - Borreguero Calvo, Jose

AU - Byrnes, James

AU - Williams, Alexis N.

AU - Cornish, Peter

AU - Pingali, Sai Venkatesh

PY - 2025/9/8

Y1 - 2025/9/8

N2 - Biological macromolecular machines occupy a continuum of structural conformations to perform cellular tasks. Mapping this conformational space provides an insight into its functionality. While the cryo-electron microscopy resolution revolution has expanded our ability to characterize the conformational continuums, there are obstacles in structurally characterizing regions of high flexibility. These technical barriers have impeded characterization of flexible ribosomal proteins when the ribosome is interacting with mRNA stem-loop structures such as a frameshifting stimulatory sequence (FSS). Small-angle neutron/X-ray scattering and electron microscopy were used to study ribosomal samples and compared structural differences between a ribosome that is bound to an FSS stem-loop compared to a ribosome bound to linear mRNA. This comparison shows that a large protein stalk elongates by 22% when the 70S interacts with an mRNA stem-loop. Our results suggest that ribosomal proteins have extensive flexibility and may influence important ribosomal mechanisms, such as those that involve FSS.

AB - Biological macromolecular machines occupy a continuum of structural conformations to perform cellular tasks. Mapping this conformational space provides an insight into its functionality. While the cryo-electron microscopy resolution revolution has expanded our ability to characterize the conformational continuums, there are obstacles in structurally characterizing regions of high flexibility. These technical barriers have impeded characterization of flexible ribosomal proteins when the ribosome is interacting with mRNA stem-loop structures such as a frameshifting stimulatory sequence (FSS). Small-angle neutron/X-ray scattering and electron microscopy were used to study ribosomal samples and compared structural differences between a ribosome that is bound to an FSS stem-loop compared to a ribosome bound to linear mRNA. This comparison shows that a large protein stalk elongates by 22% when the 70S interacts with an mRNA stem-loop. Our results suggest that ribosomal proteins have extensive flexibility and may influence important ribosomal mechanisms, such as those that involve FSS.

UR - https://www.scopus.com/pages/publications/105015535304

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JO - Biomacromolecules

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Frameshifting Stimulatory Sequence Induces Large Structural Change of Ribosomal Proteins When Bound to E. coli Ribosomes

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