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WFU Physics Colloquium

TITLE: Mechanism of action and physiological function of DbpA: a ribosomal RNA chaperon

SPEAKER: Professor Eda Koculi,

Department of Chemistry
University of Central Florida
Orlando, Florida

TIME: Wednesday January 18, 2017 at 4:00 PM

PLACE: Room 101 Olin Physical Laboratory


Refreshments will be served at 3:30 PM in the Olin Lounge. All interested persons are cordially invited to attend.

ABSTRACT

DbpA is an ATP-dependent RNA helicase whose unwinding mechanism has been has been suggested to play a fundamental role in the assembly mechanism of the 50S large ribosomal subunit. DbpA is comprised of 1) two N-terminal RecA-like domains, which perform the RNA unwinding, and 2) a C-terminal RNA-binding domain that mediates specific tethering to hairpin 92 of the Escherichia coli 23S ribosomal RNA. Hairpin 92 is located near the site of peptide bond formation, which is the most conserved region of the ribosome. Previous studies indicated that model substrates containing hairpin 92 supported the DbpA protein’s double helix unwinding activity if the double helix had a 3’ end single-stranded region, which was suggested to be the start site of the DbpA catalytic core double helix unwinding. However, our data demonstrate that the 3’ end single-stranded region is not required for the DbpA protein’s unwinding activity and that the DbpA protein unwinds a double-helix by directly loading onto it. This conclusion has important implications for the role of DbpA during ribosome assembly in vivo. It suggests that during the ribosome assembly process, DbpA could unwind substrates, which are separated from hairpin 92 by space or other macromolecules, by directly loading on those substrates. Moreover, previous studies showed that the expression of helicase inactive R331A DbpA mutant produced the accumulation of a large subunit intermediate particle. We identified two novel large subunit intermediates accumulated in cells expressing R331A DbpA. In addition, we characterized the intermediates structures and kinetics of conversion to 50S large subunit. Our experiments demonstrate that 1) the intermediates belong to three different stages of the large subunit ribosome assembly, and 2) they rearrange to form the 50S large subunit via three parallel pathways. This is the first time that the existence of multiple pathways of large subunit ribosome assembly was observed experimentally.



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