Bacteriophage-encoded 24B_1 molecule resembles herpesviral microRNAs and plays a crucial role in the development of both the virus and its host.

The 24B_1 small non-coding RNA molecule has been identified in Escherichia coli after induction of Shiga toxin-converting bacteriophage Φ24B. In this work, we focused on its direct role during phage and bacterial host development. We observed that in many aspects, this phage sRNA resembles herpesviral microRNAs.

Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of ProQ.

RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and noncoding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in , though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of in vivo and in vitro binding assays to confirm key structural features of ProQ's FinO domain and explore its mechanism of RNA interactions.

Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of ProQ.

RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and non-coding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in , though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of and binding assays to confirm key structural features of ProQ's FinO domain and explore its mechanism of RNA interactions.

KH domain proteins: Another family of bacterial RNA matchmakers?

In many bacteria, the stabilities and functions of small regulatory RNAs (sRNAs) that act by base pairing with target RNAs most often are dependent on Hfq or ProQ/FinO-domain proteins, two classes of RNA chaperone proteins. However, while all bacteria appear to have sRNAs, many have neither Hfq nor ProQ/FinO-domain proteins raising the question of whether another factor might act as an sRNA chaperone in these organisms. Several recent studies have reported that KH domain proteins, such as KhpA and KhpB, bind sRNAs.

Bacterial Chaperone Protein Hfq Facilitates the Annealing of Sponge RNAs to Small Regulatory RNAs.

Bacterial small RNAs (sRNAs) in association with the chaperone protein Hfq regulate the expression of many target mRNAs. Since sRNAs' action is crucial to engendering a response to changing environmental conditions, their activity needs to be regulated. One such mechanism occurs at the post-transcriptional level and involves sponge RNAs, which sequester sRNAs affecting their regulatory output.

Determinants of RNA recognition by the FinO domain of the Escherichia coli ProQ protein.

The regulation of gene expression by small RNAs in Escherichia coli depends on RNA binding proteins Hfq and ProQ, which bind mostly distinct RNA pools. To understand how ProQ discriminates between RNA substrates, we compared its binding to six different RNA molecules. Full-length ProQ bound all six RNAs similarly, while the isolated N-terminal FinO domain (NTD) of ProQ specifically recognized RNAs with Rho-independent terminators.

The binding of Class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs.

MgrR is an Hfq-dependent sRNA, whose transcription is controlled by the level of Mg ions in MgrR belongs to Class II sRNAs because its stability in the cell is affected by mutations in Hfq differently than canonical, Class I sRNAs. Here, we examined the effect of mutations in RNA binding sites of Hfq on the kinetics of the annealing of MgrR to two different target mRNAs, and , by global data fitting of the reaction kinetics monitored by gel electrophoresis of intermediates and products. The data showed that the mutation on the rim of the Hfq ring trapped MgrR on Hfq preventing the annealing of MgrR to either mRNA.

ProQ/FinO-domain proteins: another ubiquitous family of RNA matchmakers?

Small RNAs (sRNAs), particularly those that act by limited base pairing with mRNAs, are part of most regulatory networks in bacteria. In many cases, the base-pairing interaction is facilitated by the RNA chaperone Hfq. However, not all bacteria encode Hfq and some base-pairing sRNAs do not require Hfq raising the possibility of other RNA chaperones.

The structure of fadL mRNA and its interactions with RybB sRNA.

Small bacterial RNAs (sRNAs) regulate translation by pairing with complementary sequences in their target mRNAs, in a process which is often dependent on the Hfq protein. Here, the secondary structure of a 95-nt long fragment of Salmonella fadL mRNA containing RybB sRNA binding site in the coding region was analyzed. The data indicated local rearrangements in this mRNA structure after the annealing of RybB.

Contributions of the Hfq protein to translation regulation by small noncoding RNAs binding to the mRNA coding sequence.

The bacterial Sm-like protein Hfq affects the regulation of translation by small noncoding RNAs (sRNAs). In this way, Hfq participates in the cell adaptation to environmental stress, regulation of cellular metabolism, and bacterial virulence. The majority of known sRNAs bind complementary sequences in the 5'-untranslated mRNA regions.

Analysis of ribosomal inter-subunit sites as targets for complementary oligonucleotides.

The bacterial ribosome has many functional ribosomal RNA (rRNA) sites. We have computationally analyzed the rRNA regions involved in the interactions between the 30S and 50S subunits. Various properties of rRNA such as solvent accessibility, opening energy, hydrogen bonding pattern, van der Waals energy, thermodynamic stability were determined.

Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure.

The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5'-untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence.

Structure of bacterial regulatory RNAs determines their performance in competition for the chaperone protein Hfq.

Bacterial regulatory RNAs require the chaperone protein Hfq to enable their pairing to mRNAs. Recent data showed that there is a hierarchy among sRNAs in the competition for access to Hfq, which could be important for the tuning of sRNA-dependent translation regulation. Here, seven structurally different sRNAs were compared using filter-based competition assays.

Despite similar binding to the Hfq protein regulatory RNAs widely differ in their competition performance.

The binding of nine noncoding regulatory RNAs (sRNAs) to the E. coli Hfq protein was compared using a high-throughput double filter retention assay. Despite the fact that these sRNAs have different lengths, sequences and secondary structures their Hfq binding affinities were surprisingly uniform.

A sequence element that tunes Escherichia coli tRNA(Ala)(GGC) to ensure accurate decoding.

Mutating the rare A32-U38 nucleotide pair at the top of the anticodon loop of Escherichia coli tRNA(Ala)(GGC) to a more common U32-A38 pair results in a tRNA that performs almost normally on cognate codons but is unusually efficient in reading near-cognate codons. Pre-steady state kinetic measurements on E. coli ribosomes show that, unlike the wild-type tRNA(Ala)(GGC), the misreading mutant tRNA(Ala)(GGC) shows rapid GTP hydrolysis and no detectable proofreading on near-cognate codons.

Specificity of the ribosomal A site for aminoacyl-tRNAs.

Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2'-deoxynucleotide-substituted mRNAs are used to demonstrate the presence of a lower threshold in k(off) values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed.

tRNA residues that have coevolved with their anticodon to ensure uniform and accurate codon recognition.

The structure, phylogeny and in vivo function of the base pair formed between nucleotides 32 and 38 of the tRNA anticodon loop are reviewed. The A32-U38 pair, which is highly conserved in tRNA2(Ala) and sometimes observed in tRNA2(Pro), was recently found to decrease the affinity of tRNAs to the ribosomal A site relative to other 32-38 combinations. This suggests that the role of 32-38 pair is to tune the tRNA affinity in the A site to a uniform value.

Quantitative analysis of deoxynucleotide substitutions in the codon-anticodon helix.

The role of 2' hydroxyl groups in the codon-anticodon helix was evaluated by introducing single deoxynucleotides into each of the six positions in the helix and measuring the affinity of tRNA to either the A site or the P site of Escherichia coli 70S ribosomes. In perfect agreement with the X-ray structure of the Thermus thermophilus 30S subunit, A site binding was weaker in five of the six positions but P site binding was unaffected. Since the addition of paromomycin restores A site binding, it appears that the deoxynucleotide substituted complexes are impaired in their ability to promote the ribosomal conformational change that accompanies tRNA binding.

Idiosyncratic tuning of tRNAs to achieve uniform ribosome binding.

The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes was substantially impaired, as compared with the binding of their natural tRNAs, when they were transplanted into tRNA(2)(Ala). An analysis of chimeras composed of tRNA(2)(Ala) and various amounts of either tRNA(3)(Gly) or tRNA(2)(Arg) indicates that the presence of the parental 32-38 nucleotide pair is sufficient to restore ribosome binding of the transplanted anticodons. Furthermore, mutagenesis of tRNA(2)(Ala) showed that its highly conserved A32-U38 pair serves to weaken ribosome affinity.

The apical loop of the HIV-1 TAR RNA hairpin is stabilized by a cross-loop base pair.

The TAR hairpin of the HIV-1 RNA genome is indispensable for trans-activation of the viral promoter and virus replication. The TAR structure has been studied extensively, but most attention has been directed at the three-nucleotide bulge that constitutes the binding site of the viral Tat protein. In contrast, the conformational properties of the apical loop have remained elusive.

The bulge region of HIV-1 TAR RNA binds metal ions in solution.

Binding of Mg2+, Ca2+ and Co(NH3)6(3+) ions to the HIV-1 TAR RNA in solution was analysed by 19F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for 19F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg2+ and Ca2+ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K(d)) of 0.