Wednesday, December 31, 2025 12/31/2025

The role of collisions in rescuing stalled ribosomes in bacteria

Award Number: R01GM136960
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 02/15/2021
PERIOD OF PERFORMANCE END DATE: 12/31/2028

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The role of collisions in rescuing stalled ribosomes in bacteria - PROJECT SUMMARY Translating ribosomes often encounter obstacles that stop them in their tracks. Ribosome quality control (RQC) pathways remove stalled ribosomes from mRNA so that they can be recycled. In the absence of RQC, bacteria are hypersensitive to many clinically important antibiotics that target the ribosome. The goal of this proposal is to determine molecular mechanisms of RQC in bacteria. In the last funding cycle, we established a new paradigm for how RQC factors in bacteria selectively recognize stalled ribosomes. When a trailing ribosome collides with a stalled ribosome, a unique interface forms between them, as revealed in cryo-EM structures of collided disomes from E. coli and B. subtilis solved in collaboration with Roland Beckmann. In E. coli, SmrB binds between collided disomes and cleaves the mRNA, targeting it for degradation. Any trailing ribosomes on the truncated mRNA are quickly rescued by the well-characterized tmRNA pathway. In B. subtilis, the SmrB homolog MutS2 lacks nuclease activity and instead uses ATP hydrolysis to split stalled ribosomes into subunits. Here we address three questions raised by our discovery that ribosome collisions trigger these two different RQC pathways in E. coli and B. subtilis. First, what advantage does splitting ribosomes into subunits have compared to rescuing them with tmRNA? We hypothesize that splitting is particularly important when ribosomes are inhibited by antibiotics, given that the tmRNA pathway requires active translation. In Aim 1, we will determine how E. coli cells split stalled ribosomes, building on our preliminary data identifying a novel RQC factor with ATPase activity. We will characterize this protein biochemically and observe its activity in vivo with ribosome profiling. Second, why does SmrB cleave mRNA in E. coli whereas MutS2 in B. subtilis does not? Our preliminary data suggest that mRNAs lacking a stop codon are rapidly decayed in E. coli but not in B. subtilis. Using sequencing approaches, in Aim 2 we will compare the effects of RQC factors on the transcriptome in E. coli and B. subtilis. In addition, we will use selective ribosome profiling to identify endogenous substrates for SmrB. Finally, ribosome collisions in eukaryotes lead to the activation of signaling pathways. Are there also regulatory events that take place on collided disomes in bacteria? In disome structures, the bL9 protein from the lead ribosome reaches back and contacts the trailing ribosome. By preventing the trailing ribosome from binding EFG and attempting translocation, bL9 may prevent frameshifting. In E. coli, bL9 is phosphorylated at sites that contact the trailing ribosome; we will test whether phosphorylation of bL9 changes its activity, if phosphorylation changes under various conditions, and identify the kinase responsible. These experiments will delineate molecular mechanisms of RQC pathways that rescue stalled ribosomes in bacteria.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $300,787 )
2026	2026	THE JOHNS HOPKINS UNIVERSITY	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	000	6	12/11/2025	NON-COMPETING CONTINUATION	$300,787
														Subtotal = $300,787

Issue Date FY: 2025 ( Subtotal = $333,669 )
2025	2025	THE JOHNS HOPKINS UNIVERSITY	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	002	5	5/15/2025	COMPETING CONTINUATION	$78,197
2025	2025	THE JOHNS HOPKINS UNIVERSITY	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	000	5	12/13/2024	COMPETING CONTINUATION	$333,669
2025	2024	THE JOHNS HOPKINS UNIVERSITY	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	000	5	12/13/2024	COMPETING CONTINUATION	$0
2025	2024	THE JOHNS HOPKINS UNIVERSITY	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	001	4	5/15/2025	NON-COMPETING CONTINUATION	-$78,197
														Subtotal = $333,669

Issue Date FY: 2024 ( Subtotal = $311,688 )
2024	2024	THE JOHNS HOPKINS UNIVERSITY	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	000	4	11/7/2023	NON-COMPETING CONTINUATION	$280,519
2024	2024	THE JOHNS HOPKINS UNIVERSITY	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	001	4	8/16/2024	NON-COMPETING CONTINUATION	$31,169
														Subtotal = $311,688

Issue Date FY: 2023 ( Subtotal = $311,688 )
2023	2023	JOHNS HOPKINS UNIVERSITY, THE	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	000	3	11/18/2022	NON-COMPETING CONTINUATION	$280,519
2023	2023	JOHNS HOPKINS UNIVERSITY, THE	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	001	3	3/10/2023	NON-COMPETING CONTINUATION	$31,169
														Subtotal = $311,688

Issue Date FY: 2022 ( Subtotal = $311,688 )
2022	2022	JOHNS HOPKINS UNIVERSITY, THE	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	000	2	11/19/2021	NON-COMPETING CONTINUATION	$280,519
2022	2022	JOHNS HOPKINS UNIVERSITY, THE	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	001	2	7/28/2022	NON-COMPETING CONTINUATION	$31,169
														Subtotal = $311,688

Issue Date FY: 2021 ( Subtotal = $327,625 )
2021	2021	JOHNS HOPKINS UNIVERSITY, THE	3400 N CHARLES ST	BALTIMORE	MD	21218	BALTIMORE CITY	USA	Biomedical Research and Research Training	000	1	2/15/2021	NEW	$327,625
														Subtotal = $327,625

Grand Total All Awards = $1,897,145

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The role of collisions in rescuing stalled ribosomes in bacteria

Award Number: R01GM136960

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 02/15/2021

PERIOD OF PERFORMANCE END DATE: 12/31/2028

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