For the first time, we demonstrate directly a stable complex between a bacterial DnaG (primase) and DnaB (helicase). Utilizing fragments of both proteins, we are able to dissect interactions within this complex and provide direct evidence that it is the C-terminal domain of primase that interacts with DnaB. Furthermore, this C-terminal domain is sufficient to induce maximal stimulation of the helicase and ATPase activities of DnaB. However, the region of DnaB that interacts with the C-terminal domain of primase appears to comprise a surface on DnaB that includes regions from both of the previously identified N- and C-terminal domains. Using a combination of biochemical and physical techniques, we show that the helicase-primase complex comprises one DnaB hexamer and either two or three molecules of DnaG. Our results show that in Bacillus stearothermophilus the helicase-primase interaction at the replication fork may not be transient, as was shown to be the case in Escherichia coli. Instead, primase appears to interact with the helicase forming a tighter complex with enhanced ATPase and helicase activities.

RNA pseudouridine synthase, TruB, catalyzes pseudouridine formation at U55 in tRNA. This posttranscriptional modification is almost universally conserved and occurs in the T arm of most tRNAs. We determined the crystal structure of Escherichia coli TruB apo enzyme, as well as the structure of Thermotoga maritima TruB in complex with RNA. Comparison of the RNA-free and -bound forms of TruB reveals that this enzyme undergoes significant conformational changes on binding to its substrate. These conformational changes include the ordering of the ''thumb loop,'' which binds right into the RNA hairpin loop, and a 10°hinge movement of the C-terminal domain. Along with the result of docking experiments performed on apo TruB, we conclude that TruB recognizes its RNA substrate through a combination of rigid docking and induced fit, with TruB first rigidly binding to its target and then maximizing the interaction by induced fit. RNA modification is a posttranscriptional process whereby certain nucleotides are altered after their initial incorporation into an RNA chain. Pseudouridine (⌿) is the most abundantly found modification in RNA (1). It is found in most RNAs, including transfer, ribosomal, transfer-messenger, small nuclear, and small nucleolar guide RNAs. Many ⌿ residues are highly conserved and appear to be confined to the functionally important part of RNA. For example, ⌿s are clustered within the peptidyl transferase center of the ribosome (2), are conserved within regions of small nuclear RNAs that are involved in RNA-RNA interactions (3), and have been implicated in spliceosome assembly (4).The most obvious structural effect of ⌿ formation is the creation of a new hydrogen bond donor N-H, located where C5 used to be. It has been shown that pseudouridylation has the effect of enhancing local RNA stacking in both single-stranded and duplex regions, resulting in increased conformational stability (5, 6). Certain genetic mutants lacking specific ⌿ residues in tRNA or rRNA exhibit difficulties in translation, display slow growth rates, and fail to compete effectively with wild-type strains in mixed culture (7-10). All of the evidence indicates that ⌿s play an important and critical role in RNA-mediated cellular processes. The precise role of this modification, however, remains unclear.⌿ synthases catalyze the isomerization of U to ⌿. The general mechanism for these enzymes requires a nucleophilic attack on C6 of the uracil ring in the target U by a universally conserved Asp residue, which leads to the breakage of the glycosidic bond, followed by a rotation of the uracil ring and reattachment of the C5 atom of the uracil to C1Ј of the ribose (Fig. 1A) (11). In prokaryotes, pseudouridylation is mediated by a set of enzymes that are site-or region-specific; each of these enzymes specifies the formation of just one or sometimes several ⌿s in RNA. Although the reaction catalyzed by each of these enzymes is the same, the substrate specificity varies from simple stem-loop structures to larger and more complex RNA.TruB catal...

No abstract

Hydrogel-based self-healing ionic skins possess high mechanical strength, excellent resilience, anti-freezing properties and high sensitivity and can heal fatigue and mechanical damage to restore the original sensing performance.