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.

DbpA is a DEAD-box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA-like catalytic core responsible for double-helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core’s grasp. In other words, it is not known if DbpA is a site-specific enzyme or region-specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region-specific or a site-specific enzyme. Our data suggest that DbpA is a region-specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C-terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double-helices in its vicinity. The only requirement for a double-helix to serve as a DbpA substrate is for the double-helix to be positioned within the catalytic core’s grasp.

mutant derivatives are investigated with optical tweezers, steered molecular dynamics (SMD) simulations and single-molecule FRET (smFRET). With smFRET, we show that base triples at one end (formed by loop 1 and stem 2) of hTR-PK promote the formation of those at the distal end (formed by stem 1 and loop 2), and thereby limit the flexibility of the distal loop (loop 2). The coordination between base triples is therefore responsible for the single-step unfolding event of the structure. By contrast, smFRET experiments identify a compact intermediate structure before hTR-PK is completely disrupted by the ribosome. SMD simulations further reveal that as the hTR-PK loop 2 attaches to the positively charged residues of ribosomal protein S3, base triples facilitate formation of the unfolding intermediate of hTR-PK. When the base triples are disrupted by mutations (the delta-triple mutant), the compact unfolding intermediate can no longer be seen in SMD simulations and smFRET experiments (that is, the mutant exhibits single-step unfolding). The delta-triple mutation also results in a dramatic drop in FS efficiency from 50% to 0%. Our study demonstrates the importance of a base triple-stabilized unfolding intermediate in PK-induced FS.

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