Biomedical subjects

A K Amarasinghe

Publications and source records attributed to A K Amarasinghe.

4 recordsLinked to original sources

PubMed2001

Ethidium-dependent uncoupling of substrate binding and cleavage by Escherichia coli ribonuclease III.

Ethidium bromide (EB) is known to inhibit cleavage of bacterial rRNA precursors by Escherichia coli ribonuclease III, a dsRNA-specific nuclease. The mechanism of EB inhibition of RNase III is not known nor is there information on EB-binding sites in RNase III substrates. We show here that EB is a reversible, apparently competitive inhibitor of RNase III cleavage of small model substrates in vitro. Inhibition is due to intercalation, since (i) the inhibitory concentrations of EB are similar to measured EB intercalation affinities; (ii) substrate cleavage is not affected by actinomycin D, an intercalating agent that does not bind dsRNA; (iii) the EB concentration dependence of inhibition is a function of substrate structure. In contrast, EB does not strongly inhibit the ability of RNase III to bind substrate. EB also does not block substrate binding by the C-terminal dsRNA-binding domain (dsRBD) of RNase III, indicating that EB perturbs substrate recognition by the N-terminal catalytic domain. Laser photocleavage experiments revealed two ethidium-binding sites in the substrate R1.1 RNA. One site is in the internal loop, adjacent to the scissile bond, while the second site is in the lower stem. Both sites consist of an A-A pair stacked on a CG pair, a motif which apparently provides a particularly favorable environment for intercalation. These results indicate an inhibitory mechanism in which EB site-specifically binds substrate, creating a cleavage-resistant complex that can compete with free substrate for RNase III. This study also shows that RNase III recognition and cleavage of substrate can be uncoupled and supports an enzymatic mechanism of dsRNA cleavage involving cooperative but not obligatorily linked actions of the dsRBD and the catalytic domain.

Base Sequence↗

PubMed2001

Escherichia coli ribonuclease III: affinity purification of hexahistidine-tagged enzyme and assays for substrate binding and cleavage.

It is now evident that members of the RNase III family of nucleases have central roles in prokaryotic and eukaryotic RNA maturation and decay pathways. Ongoing research is uncovering new roles for RNase III homologs. For example, the phenomena of RNA interference (RNAi) and posttranscriptional gene silencing (PTGS) involve dsRNA processing, carried out by an RNase III homolog. We anticipate an increased focus on the mechanism, regulation, and biological roles of RNase III orthologs. Although the differences in the physicochemical properties of RNase III orthologs, and distinct substrate reactivity epitopes and ionic requirements for optimal activity, may mean that the protocols describe here are not strictly transferrable, the affinity purification methodology, and substrate preparation and use should be generally applicable.

Base Sequence↗

PubMed2001

An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element.

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5' exon. This element contains two pseudo-5' splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5' splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5' exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5' splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro.

Animals↗

PubMed1994

Salmonella typhi endocarditis.

We report the case history of a patient with Salmonella typhi endocarditis. Bacteriological diagnosis was made by bone marrow culture when isolation from blood was unsuccessful. This is the first case of S typhi endocarditis to be documented in Sri Lanka.

Adult↗