TY - JOUR
T1 - Mutational characterization and mapping of the 70S ribosome active site
AU - D'Aquino, Anne E.
AU - Azim, Tasfia
AU - Aleksashin, Nikolay A.
AU - Hockenberry, Adam J.
AU - Krüger, Antje
AU - Jewett, Michael C.
N1 - Funding Information:
Army Research Office Grant [W911NF-18-1-0181 and W911NF-16-1-0372 to M.C.J.]; National Science Foundation Grant [MCB-1716766 to M.C.J.]; Northwestern University Biophysics Training Program (to A.E.D., in part); Northwestern University Ryan Fellowship (to A.E.D); Northwestern University Presidential Fellowship (to A.E.D.); National Science Foundation Graduate Research Fellowship Program (to A.E.D); M.C.J also acknowledges the David and Lucile Packard Foundation and the Camille Dreyfus Teacher-Scholar Program. Funding for open access charge: Army Research Office [W911NF-16-1-0372].
Publisher Copyright:
© 2020 The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2020/3/18
Y1 - 2020/3/18
N2 - The synthetic capability of the Escherichia coli ribosome has attracted efforts to repurpose it for novel functions, such as the synthesis of polymers containing non-natural building blocks. However, efforts to repurpose ribosomes are limited by the lack of complete peptidyl transferase center (PTC) active site mutational analyses to inform design. To address this limitation, we leverage an in vitro ribosome synthesis platform to build and test every possible single nucleotide mutation within the PTC-ring, A-loop and P-loop, 180 total point mutations. These mutant ribosomes were characterized by assessing bulk protein synthesis kinetics, readthrough, assembly, and structure mapping. Despite the highly-conserved nature of the PTC, we found that >85% of the PTC nucleotides possess mutational flexibility. Our work represents a comprehensive single-point mutant characterization and mapping of the 70S ribosome's active site. We anticipate that it will facilitate structure-function relationships within the ribosome and make possible new synthetic biology applications.
AB - The synthetic capability of the Escherichia coli ribosome has attracted efforts to repurpose it for novel functions, such as the synthesis of polymers containing non-natural building blocks. However, efforts to repurpose ribosomes are limited by the lack of complete peptidyl transferase center (PTC) active site mutational analyses to inform design. To address this limitation, we leverage an in vitro ribosome synthesis platform to build and test every possible single nucleotide mutation within the PTC-ring, A-loop and P-loop, 180 total point mutations. These mutant ribosomes were characterized by assessing bulk protein synthesis kinetics, readthrough, assembly, and structure mapping. Despite the highly-conserved nature of the PTC, we found that >85% of the PTC nucleotides possess mutational flexibility. Our work represents a comprehensive single-point mutant characterization and mapping of the 70S ribosome's active site. We anticipate that it will facilitate structure-function relationships within the ribosome and make possible new synthetic biology applications.
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U2 - 10.1093/nar/gkaa001
DO - 10.1093/nar/gkaa001
M3 - Article
C2 - 32009164
AN - SCOPUS:85080994599
VL - 48
SP - 2777
EP - 2789
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 5
ER -