Characterization of the Structure and Melting Behavior of the Loop I fragment of ColE1 RNA I

Abstract

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5′-end, and a 31-mer which has 11 5′-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5′ end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32°C and 71°C clearly discernible. In conjunction with NMR melting study the major transition at 71°C is assigned to the overall melting of the stem region and the 32°C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.     

Probing the substrate specificity of Escherichia coli RNase E using a novel oligonucleotide-based assay

Endoribonuclease RNase E has a central role in both processing and decay of RNA in Escherichia coli, and apparently in many other organisms, where RNase E homologs were identified or their existence has been predicted from genomic data. Although the biochemical properties of this enzyme have been already studied for many years, the substrate specificity of RNase E is still poorly characterized. Here, I have described a novel oligonucleotide-based assay to identify specific sequence determinants that either facilitate or impede the recognition and cleavage of RNA by the catalytic domain of the enzyme. The knowledge of these determinants is crucial for understanding the nature of RNA–protein interactions that control the specificity and efficiency of RNase E cleavage and opens new perspectives for further studies of this multi-domain protein. Moreover, the simplicity and efficiency of the proposed assay suggest that it can be a valuable tool not only for the characterization of RNase E homologs but also for the analysis of other site-specific nucleases.     

Translation initiation and the fate of bacterial mRNAs

Studies in pro- and eukaryotes have revealed that translation can determine the stability of a given messenger RNA. In bacteria, intrinsic mRNA signals can confer efficient ribosome binding, whereas translational feedback inhibition or environmental cues can interfere with this process. Such regulatory mechanisms are often controlled by RNA-binding proteins, small noncoding RNAs and structural rearrangements within the 5' untranslated region. Here, we review molecular events occurring in the 5' untranslated region of primarily Escherichia coli mRNAs with regard to their effects on mRNA stability.     

Distinct and overlapping binding sites of Pseudomonas aeruginosa Hfq and RsmA proteins on the non-coding RNA RsmY

In Pseudomonas aeruginosa the Rsm system is involved in regulation of quorum-sensing and virulence gene expression. Our recent studies revealed that the stability and abundance of the non-coding RNA RsmY, which antagonizes the translational regulator RsmA, is dependent on Hfq. Here, we show that Hfq and RsmA bind concurrently to RsmY. Enzymatic probing of RsmY RNA in the presence of RsmA and Hfq verified the proposed -GGA- motifs as RsmA binding sites and located Hfq binding sites in single-stranded regions adjacent to stem-loop structures, respectively. We conclude that distinct binding of Hfq and RsmA on RsmY RNA permits RsmY-mediated RsmA titration upon binding to and stabilization of RsmY RNA by Hfq. In addition, we provide evidence that Hfq sequesters RNase E cleavage sites on RsmY, which explains the previously observed dependence of RsmY RNA stability on Hfq.     

Functional effects of variants of the RNA chaperone Hfq

The ring-shaped RNA chaperone Hfq has recently received much attention owing to its multiple roles in RNA metabolism. In this study we have performed a mutational analysis of the Escherichia coli hfq gene, and have studied the effects of amino acid substitutions at several positions in the Hfq protein as well as of C-terminal truncations on its role in phage Qbeta replication, in repression of a target mRNA, and on the stability of the small regulatory RNA DsrA. These functional studies provided insights into the interaction of Hfq with RNA and suggested a role for the C-terminus of Hfq in DsrA stability.     

Immunohistochemical expression of rabphilin-3A-like (Noc2) in normal and tumor tissues of human endocrine pancreas

Involvement of rabphilin-3A-like (RPH3AL), or Noc2, the potential effector of Ras-associated binding proteins Rab3A and Rab27A in the regulation of exocytotic processes in the endocrine pancreas has been demonstrated in experimental models. Noc2 expression together with other regulatory molecules of the exocytotic machinery in human tissues, however, has not been studied. We evaluated immunohistochemical expression of the key molecules of the exocytotic machinery, Noc2, Rab3A, Rab27A, and RIM2, together with the characteristic islet cell hormones, insulin and glucagon in normal and endocrine tumor tissues of human pancreas. Normal pancreatic islets were stained for all of these proteins and showed strong cytoplasmic localization. A similar pattern of strong cytoplasmic expression of these proteins was observed in the majority of endocrine tumors. By contrast, the exocrine portions of normal appearing pancreas completely lacked Rab27A staining and showed decreased expression of the proteins, Noc2, Rab3A, and RIM2. The staining pattern of Noc2 and Rab27A was similar to the staining pattern of glucagon-producing cells within the islets. The concomitant expression of Noc2 with these molecules suggests that Noc2 may serve as an effector for Rab3A and Rab27A and that it is involved in the regulation of exocytosis of the endocrine pancreas in humans.