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Structural and biochemical characterization of ribosomal RNA methylation by radical SAM enzymes

Award Number: F32GM163367
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 02/20/2026
PERIOD OF PERFORMANCE END DATE: 02/19/2029

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Structural and biochemical characterization of ribosomal RNA methylation by radical SAM enzymes - Project Summary Antibiotic resistance is a major global health challenge, leading to 1.14 million deaths worldwide and 2.8 million infections annually in the United States. Infection by multidrug-resistant strains such as methicillin- resistant Staphylococcus aureus have grown by 100% since 1990 and are primarily responsible for these statistics. These pathogens employ myriad strategies to resist antibiotics, including modifying macromolecules that antibiotics target within bacterial cells. One such strategy is the methylation of adenosine 2503 (A2503) of 23S ribosomal RNA by the enzyme Cfr which confers resistance to several classes of antibiotics. A2503 is also methylated by RlmN in Escherichia coli which enhances translational fidelity. These enzymes belong to the radical S-adenosylmethionine (SAM) superfamily of enzymes which contain at least one iron-sulfur (Fe-S) cluster. Both enzymes utilize a methyl-cysteine radical to active the substrate, generating a protein-RNA cross- linked (PRCL) species which undergoes deprotonation and radical fragmentation to yield the methylated product. RlmN methylates C2 of A2503 in the 23S rRNA and A37 various tRNA molecules, making it one of only two known RNA-modifying enzymes with dual specificity. Cfr preferentially modifies rRNA at C8 but can also methylate C2 to generate dimethyladenosine. The project goal is to understand the basis of dual specificity methylation by RlmN and site-selectivity of Cfr. Previous work has demonstrated that mutation of an active-site cysteine results in a stable PRCL species suitable for structural and spectroscopic characterization. The structure of Cfr modifying C8 of an 87-mer rRNA substrate has recently been solved by cryo-EM. We will determine the structure of Cfr modifying C2 by enzymatic synthesis of 8-methyl-adenosine 87-mer (m8A) followed by cryo-EM of the PRCL. This will reveal the basis for site-selectivity and dimethylation activity in this enzyme. The structure of RlmN bound to tRNA has been solved by X-ray crystallography. We propose to solve the structure of RlmN bound to the 87-mer rRNA using cryo-EM. Comparison to the tRNA-bound structure will uncover the basis of the dual specificity will be followed by mutagenesis of Cfr to test these hypotheses. The C8-bound PRCL radical species has been observed in Cfr by EPR spectroscopy but never in RlmN. We will characterize the C2-bound Cfr species by EPR and ENDOR using the m8A substrate to determine if this kinetic behavior is due to the protein environment or the site of modification. These studies will yield fundamental insights into both catalysis and antibiotic resistance mechanisms. This work will be carried out in the laboratory of Prof. Squire J. Booker at the University of Pennsylvania (Penn) and involve collaborations at Penn State University and Northwestern University. Prof. Booker is the preeminent figure in the field of radical SAM enzymology and his laboratory is highly experienced in the proposed methods, making this an ideal training environment. The project will incorporate training in cryo-EM, anaerobic biochemistry, RNA biology, spectroscopy, and kinetics.


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 = $76,300 )
2026	2026	TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, THE	3451 WALNUT STREET	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Biomedical Research and Research Training	000	1	2/20/2026	NEW	$75,052
2026	2026	TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, THE	3451 WALNUT ST	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Biomedical Research and Research Training	002	1	5/11/2026	NEW	$1,248
2026	2026	TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, THE	3451 WALNUT ST	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Biomedical Research and Research Training	001	1	2/20/2026	NEW	$0
														Subtotal = $76,300

Grand Total All Awards = $76,300

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Structural and biochemical characterization of ribosomal RNA methylation by radical SAM enzymes

Award Number: F32GM163367

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 02/20/2026

PERIOD OF PERFORMANCE END DATE: 02/19/2029

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