DbpA is a DEAD-box RNA helicase implicated in Escherichia coli large ribosomal subunit assembly. Previous studies have shown that when the ATPase and helicase inactive DbpA construct, R331A, is expressed in E. coli cells, a large ribosomal subunit intermediate accumulates. The large subunit intermediate migrates as a 45S particle in a sucrose gradient. Here, using a number of structural and fluorescent assays, we investigate the ribosome profiles of cells lacking wild-type DbpA and overexpressing the R331A DbpA construct. Our data show that in addition to the 45S particle previously described, 27S and 35S particles are also present in the ribosome profiles of cells overexpressing R331A DbpA. The 27S, 35S, and 45S independently convert to the 50S subunit, suggesting that ribosome assembly in the presence of R331A and the absence of wild-type DbpA occurs via multiple pathways.

DbpA is a DEAD-box RNA helicase implicated in the assembly of the large ribosomal subunit. Similar to all the members of the DEAD-box family, the DbpA protein has two N-terminal RecA-like domains, which perform the RNA unwinding. However, unlike other members of this family, the DbpA protein also possesses a structured C-terminal RNA-binding domain that mediates specific tethering of DbpA to hairpin 92 of the Escherichia coli 23S ribosomal RNA. Previous studies using model RNA molecules containing hairpin 92 show that the RNA molecules support the DbpA protein's double-helix unwinding activity, provided that the double helix has a 3 ′ single-stranded region. The 3 ′ single-stranded region was suggested to be the start site of the DbpA protein's catalytic unwinding activity. The data presented here demonstrate that the single-stranded region 3′ of the doublehelix substrate is not required for the DbpA protein's unwinding activity and the DbpA protein unwinds the double-helix substrates by directly loading on them.

openAccessArticle: Falsecover date: 2015-01-27pii: S0006-3495(14)03351-7Harvest Date: 2016-01-06 13:08:07issueName:Page Range: 390a-390ahref scidir: http://www.sciencedirect.com/science/article/pii/S0006349514033517pubType