Membrane-Coupled mRNA Trafficking in Fungi.

Intracellular logistics are essential for delivery of newly synthesized material during polar growth of fungal hyphae. Proteins and lipids are actively transported throughout the cell by motor-dependent movement of small vesicles or larger units such as endosomes and the endoplasmic reticulum. A remarkably tight link is emerging between active membrane trafficking and mRNA transport, a process that determines the precise subcellular localization of translation products within the cell. Here, we report on recent insights into the mechanism and biological role of these intricate cotransport processes in fungal models such as Saccharomyces cerevisiae, Candida albicans, and Ustilago maydis. In the latter, we focus on the new finding of endosomal mRNA transport and its implications for protein targeting, complex assembly, and septin biology.

6S RNA: recent answers--future questions.

6S RNA is a non-coding RNA, found in almost all phylogenetic branches of bacteria. Through its conserved secondary structure, resembling open DNA promoters, it binds to RNA polymerase and interferes with transcription at many promoters. That way, it functions as transcriptional regulator facilitating adaptation to stationary phase conditions. Strikingly, 6S RNA acts as template for the synthesis of small RNAs (pRNA), which trigger the disintegration of the inhibitory RNA polymerase-6S RNA complex releasing 6S RNA-dependent repression. The regulatory implications of 6S RNAs vary among different bacterial species depending on the lifestyle and specific growth conditions that they have to face. The influence of 6S RNA can be seen on many different processes including stationary growth, sporulation, light adaptation or intracellular growth of pathogenic bacteria. Recent structural and functional studies have yielded details of the interaction between E. coli 6S RNA and RNA polymerase. Genome-wide transcriptome analyses provided insight into the functional diversity of 6S RNAs. Moreover, the mechanism and physiological consequences of pRNA synthesis have been explored in several systems. A major function of 6S RNA as a guardian regulating the economic use of cellular resources under limiting conditions and stress emerges as a common perception from numerous recent studies.

Regulation of transcription by 6S RNAs: insights from the Escherichia coli and Bacillus subtilis model systems.

Whereas, the majority of bacterial non-coding RNAs and functional RNA elements regulate post-transcriptional processes, either by interacting with other RNAs via base-pairing or through binding of small ligands (riboswitches), 6S RNAs affect transcription itself by binding to the housekeeping holoenzyme of RNA polymerase (RNAP). Remarkably, 6S RNAs serve as RNA templates for bacterial RNAP, giving rise to the de novo synthesis of short transcripts, termed pRNAs (product RNAs). Hence, 6S RNAs prompt the enzyme to act as an RNA-dependent RNA polymerase (RdRP). Synthesis of pRNAs exceeding a certain length limit (~13 nt) persistently rearrange the 6S RNA structure, which in turn, disrupts the 6S RNA:RNAP complex. This pRNA synthesis-mediated "reanimation" of sequestered RNAP molecules represents the conceivably fastest mechanism for resuming transcription in cells that enter a new exponential growth phase. The many different 6S RNAs found in a wide variety of bacteria do not share strong sequence homology but have in common a conserved rod-shaped structure with a large internal loop, termed the central bulge; this architecture mediates specific binding to the active site of RNAP. In this article, we summarize the overall state of knowledge as well as very recent findings on the structure, function, and physiological effects of 6S RNA examples from the two model organisms, Escherichia coli and Bacillus subtilis. Comparison of the presently known properties of 6S RNAs in the two organisms highlights common principles as well as diverse features.

6S RNA - an old issue became blue-green.

6S RNA from Escherichia coli acts as a versatile transcriptional regulator by binding to the RNA polymerase and changing promoter selectivity. Although homologous 6S RNA structures exist in a wide range of bacteria, including cyanobacteria, our knowledge of 6S RNA function results almost exclusively from studies with E. coli. To test for potential structural and functional conservation, we selected four predicted cyanobacterial 6S RNAs (Synechocystis, Synechococcus, Prochlorococcus and Nostoc), which we compared with their E. coli counterpart. Temperature-gradient gel electrophoresis revealed similar thermodynamic transition profiles for all 6S RNAs, indicating basically similar secondary structures. Subtle differences in melting behaviour of the different RNAs point to minor structural variations possibly linked to differences in optimal growth temperature. Secondary structural analysis of three cyanobacterial 6S RNAs employing limited enzymic hydrolysis and in-line probing supported the predicted high degree of secondary structure conservation. Testing for functional homology we found that all cyanobacterial 6S RNAs were active in binding E. coli RNA polymerase and transcriptional inhibition, and had the ability to act as template for transcription of product RNAs (pRNAs). Deletion of the 6S RNA gene in Synechocystis did not significantly affect cell growth in liquid media but reduced fitness during growth on solid agar. While our study shows that basic 6S RNA functions are conserved in species as distantly related as E. coli and cyanobacteria, we also noted a subtle degree of divergence, which might reflect fundamental differences in transcriptional regulation and lifestyle, thus providing the first evidence for a possible physiological role in cyanobacteria.

A conformational switch is responsible for the reversal of the 6S RNA-dependent RNA polymerase inhibition in Escherichia coli.

6S RNA is a bacterial transcriptional regulator,which accumulates during stationary phase and inhibits transcription from many promoters due to stable association with σ 70 -containing RNA polymerase. This inhibitory RNA polymerase ∼ 6S RNA complex dissociates during nutritional upshift, when cells undergo outgrowth from stationary phase, releasing active RNA polymerase ready for transcription. The release reaction depends on a characteristic property of 6S RNAs, namely to act as template for the de novo synthesis of small RNAs, termed pRNAs.Here, we used limited hydrolysis with structure-specific RNases and in-line probing of isolated 6S RNA and 6SRNA ∼ pRNA complexes to investigate the molecular details leading to the release reaction. Our results indicate that pRNA transcription induces the refolding of the 6S RNA secondary structure by disrupting part of the closing stem(conserved sequence regions CRI and CRIV) and formation of a new hairpin (conserved sequence regions CRIII and CRIV). Comparison of the dimethylsulfate modification pattern of 6S RNA in living cells at stationary growth and during outgrowth confirmed the conformational change observed in vitro. Based on our results, a model describing the individual steps of the release reaction is presented.

E. coli 6S RNA: a universal transcriptional regulator within the centre of growth adaptation.

Bacterial 6S RNA has been shown to bind with high affinity to σ(70)-containing RNA polymerase, suppressing σ(70)-dependent transcription during stationary phase, when 6S RNA concentrations are highest. We recently reported a genome-wide transcriptional comparison of wild-type and 6S RNA deficient E. coli strains. Contrary to the expected σ(70)- and stationary phase-specific regulatory effect of 6S RNA it turned out that mRNA levels derived from many alternative sigma factors, including σ(38) or σ(32), were affected during exponential and stationary growth. Among the most noticeably down-regulated genes at stationary growth are ribosomal proteins and factors involved in translation. In addition, a striking number of mRNA levels coding for enzymes involved in the purine metabolism, for transporters and stress regulators are altered both during log- and stationary phase. During the study we discovered a link between 6S RNA and the general stress alarmone ppGpp, which has a higher basal level in cells deficient in 6S RNA. This finding points to a functional interrelation of 6S RNA and the global network of stress and growth adaptation.

Mapping the spatial neighborhood of the regulatory 6S RNA bound to Escherichia coli RNA polymerase holoenzyme.

Bacterial 6S RNA interacts specifically with RNA polymerase acting as transcriptional regulator. Until now, no detailed characterization of the spatial arrangement of the non-coding RNA within the three-dimensional structure of RNA polymerase has been performed. Here we present results obtained with the chemical nuclease FeBABE tethered to distinct positions of RNA polymerase σ(70) subunit. 6S RNA complexes were formed with a collection of RNA polymerases, where the cleavage reagent had been fused to σ(70) single-cysteine variants close to regions involved in promoter recognition. FeBABE-induced cleavage sites within the 6S RNA structure were identified, indicating close spatial neighborhood between σ(70) single-cysteine side chains and defined positions of the 6S RNA structure. Our analysis demonstrates close proximity between the 6S RNA internal hairpin and σ(70) domain 4.2, normally involved in recognition of -35 promoter DNA. Defined sections of the internal 6S RNA stem structure flanking the central bubble are positioned near conserved σ(70) domains 3.1, 2.3 and 2.1, which are implicated in binding and melting DNA promoters between the -10 and -35 elements. Moreover, we show that U44 of 6S RNA is located near RNA polymerase active site (σ(70) domain 3.2), fully consistent with its function as starting nucleotide in RNA-directed pRNA transcription. No neighboring contacts were detected between 6S RNA and σ(70) region 1.2 or between σ(70) and the 6S RNA closing stem structure (residues 1-41 and 144-184). Results were used to dock a structural model of 6S RNA to the known three-dimensional structure of Escherichia coli σ(70) RNA polymerase holoenzyme.