Birgit Klinkert

Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution

In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5′-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA.

The Escherichia coli ibpA thermometer is comprised of stable and unstable structural elements

Translation of many small heat shock genes in α- and γ-proteobacteria is controlled by the ROSE (Repression Of heat Shock gene Expression) element, a thermo-responsive RNA structure in the 5’-untranslated region. ROSEibpA regulates translation of the Escherichia coli ibpA gene coding for an inclusion body-associated protein. We present first structural insights into a full-length ROSE element by examining the temperature-induced conformational changes of ROSEibpA using detailed enzymatic and lead probing experiments between 20 and 50 °C. The initial two hairpins are stable at all temperatures tested and might assist in proper folding of the third temperature-responsive stem-loop structure, which restricts access to the Shine-Dalgarno sequence at temperatures below 35 °C. Toeprinting (primer extension inhibition) experiments show that binding of the 30S ribosome to ROSEibpA is enhanced at high temperatures. In contrast to other ROSE-like elements, the final hairpin is rather short. Single point mutations result in alternative structures with positive or negative effects on translation efficiency. Our study demonstrates how the combination of stable and unstable modules controls translation efficiency in a complete RNA thermometer.

Modulation of the stability of the Salmonella fourU-type RNA thermometer

RNA thermometers are translational control elements that regulate the expression of bacterial heat shock and virulence genes. They fold into complex secondary structures that block translation at low temperatures. A temperature increase releases the ribosome binding site and thus permits translation initiation. In fourU-type RNA thermometers, the AGGA sequence of the SD region is paired with four consecutive uridines. We investigated the melting points of the wild-type and mutant sequences. It was decreased by 5°C when a stabilizing GC basepair was exchanged by an AU pair or increased by 11°C when an internal AG mismatch was converted to a GC pair, respectively. Stabilized or destabilized RNA structures are directly correlated with decreased or increased in vivo gene expression, respectively. Mg2+ also affected the melting point of the fourU thermometer. Variations of the Mg2+ concentration in the physiological range between 1 and 2 mM translated into a 2.8°C shift of the melting point. Thus, Mg2+ binding to the hairpin RNA is regulatory relevant. Applying three different NMR techniques, two Mg2+ binding sites were found in the hairpin structure. One of these binding sites could be identified as outer sphere binding site that is located within the fourU motif. Binding of the two Mg2+ ions exhibits a positive cooperativity with a Hill coefficient of 1.47. Free energy values ΔG for Mg2+ binding determined by NMR are in agreement with data determined from CD measurements.

Translation of chloroplast psbD mRNA in Chlamydomonas is controlled by a secondary RNA structure blocking the AUG start codon

Translation initiation represents a key step during regulation of gene expression in chloroplasts. Here, we report on the identification and characterization of three suppressor point mutations which overcome a translational defect caused by the deletion of a U-rich element in the 5′-untranslated region (5′-UTR) of the psbD mRNA in the green alga Chlamydomonas reinhardtii. All three suppressors affect a secondary RNA structure encompassing the psbD AUG initiation codon within a double-stranded region as judged by the analysis of site-directed chloroplast mutants as well as in vitro RNA mapping experiments using RNase H. In conclusion, the data suggest that these new element serves as a negative regulator which mediates a rapid shut-down of D2 synthesis.

Evolution from the Prokaryotic to the Higher Plant Chloroplast Signal Recognition Particle: The Signal Recognition Particle RNA Is Conserved in Plastids of a Wide Range of Photosynthetic Organisms

This article provides an analysis of chloroplast signal recognition particle (cpSRP) evolution within the green and red lineages. A focus lies on the distribution and characterization of the plastid-encoded SRP RNA component. Furthermore, the cpSRP system of Physcomitrella patens containing an SRP RNA and a cpSRP43 component was investigated, and the structure of the cpFtsY receptor was solved. The protein targeting signal recognition particle (SRP) pathway in chloroplasts of higher plants has undergone dramatic evolutionary changes. It disposed of its RNA, which is an essential SRP component in bacteria, and uses a unique chloroplast-specific protein cpSRP43. Nevertheless, homologs of the conserved SRP54 and the SRP receptor, FtsY, are present in higher plant chloroplasts. In this study, we analyzed the phylogenetic distribution of SRP components in photosynthetic organisms to elucidate the evolution of the SRP system. We identified conserved plastid SRP RNAs within all nonspermatophyte land plant lineages and in all chlorophyte branches. Furthermore, we show the simultaneous presence of cpSRP43 in these organisms. The function of this novel SRP system was biochemically and structurally characterized in the moss Physcomitrella patens. We show that P. patens chloroplast SRP (cpSRP) RNA binds cpSRP54 but has lost the ability to significantly stimulate the GTPase cycle of SRP54 and FtsY. Furthermore, the crystal structure at 1.8-Å resolution and the nucleotide specificity of P. patens cpFtsY was determined and compared with bacterial FtsY and higher plant chloroplast FtsY. Our data lead to the view that the P. patens cpSRP system occupies an intermediate position in the evolution from bacterial-type SRP to higher plant-type cpSRP system.

Constitutive production of c-di-GMP is associated with mutations in a variant of Pseudomonas aeruginosa with altered membrane composition

Changes in the fatty acid composition of the plasma membrane in an opportunistic pathogen promote biofilm formation. Making bacteria sticky When free-swimming bacteria encounter a surface, such as the inner lining of a catheter or an airway in a patient with cystic fibrosis (CF), they aggregate to form a biofilm, which is an encapsulated, multicellular structure that has increased resistance to environmental conditions and antimicrobial compounds. Blanka et al. sequenced a small colony variant of the opportunistic pathogen Pseudomonas aeruginosa, isolated from the lung of a patient with CF, and found a mutation in a gene cluster that encodes a multisubunit enzyme responsible for fatty acid biosynthesis, which altered the composition of the P. aeruginosa plasma membrane. The altered membrane composition resulted in the constitutive production of the second messenger c-di-GMP, which promotes biofilm formation. Understanding the opportunistic adaptations that promote biofilm formation may help in designing therapies to treat biofilm-associated infections. Most bacteria can form multicellular communities called biofilms on biotic and abiotic surfaces. This multicellular response to surface contact correlates with an increased resistance to various adverse environmental conditions, including those encountered during infections of the human host and exposure to antimicrobial compounds. Biofilm formation occurs when freely swimming (planktonic) cells encounter a surface, which stimulates the chemosensory-like, surface-sensing system Wsp and leads to generation of the intracellular second messenger 3′,5′-cyclic-di-guanosine monophosphate (c-di-GMP). We identified adaptive mutations in a clinical small colony variant (SCV) of Pseudomonas aeruginosa and correlated their presence with self-aggregating growth behavior and an enhanced capacity to form biofilms. We present evidence that a point mutation in the 5′ untranslated region of the accBC gene cluster, which encodes components of an enzyme responsible for fatty acid biosynthesis, was responsible for a stabilized mRNA structure that resulted in reduced translational efficiency and an increase in the proportion of short-chain fatty acids in the plasma membrane. We propose a model in which these changes in P. aeruginosa serve as a signal for the Wsp system to constitutively produce increased amounts of c-di-GMP and thus play a role in the regulation of adhesion-stimulated bacterial responses.

Relationship between mRNA levels and protein accumulation in a chloroplast promoter-mutant of Chlamydomonas reinhardtii

The photosynthetic chloroplast mutant G64 of Chlamydomonas reinhardtii was shown to contain a single point mutation within the 5′ region of the psbD gene encoding the D2 protein of the photosystem II reaction center. The mutation affects the sequence element TATAATAT which has previously been hypothesized to function as the psbD promoter. Run-on analysis confirmed that transcription of psbD in the mutant was reduced to approximately 10% of the wild-type level. However, psbD mRNA accumulated to approximately 35%, despite the prominent decrease in RNA synthesis. This suggests that RNA-stabilization effects can compensate to some extent for a reduction in transcriptional activity. Interestingly, a direct correlation between transcript levels and the accumulation of the psbD gene product, the D2-protein, was observed in G64. The data suggest that posttranscriptionally acting regulatory factors determine the rate-limiting steps of chloroplast psbD gene expression.

Translational control of small heat shock genes in mesophilic and thermophilic cyanobacteria by RNA thermometers

Cyanobacteria constitute a heterogeneous phylum of oxygen-producing, photosynthetic prokaryotes. They are susceptible to various stress conditions like heat, salt, or light stress, all inducing the cyanobacterial heat shock response (HSR). Cyanobacterial small heat shock proteins (sHsps) are known to preserve thylakoid membrane integrity under stress conditions, thereby protecting the photosynthesis machinery. In Synechocystis sp PCC 6803, synthesis of the sHsp Hsp17 is regulated by an RNA thermometer (RNAT) in the 5′-untranslated region (5′-UTR) of the hsp17 mRNA. RNATs are direct temperature sensors that control expression of many bacterial heat shock and virulence genes. They hinder translation at low temperatures by base pairing, thus blocking ribosome access to the mRNA. To explore the temperature range in which RNATs act, we studied various RNAT candidates upstream of sHsp genes from mesophilic and thermophilic cyanobacteria. The mesophilic cyanobacteria Anabaena variabilis and Nostoc sp chromosomally encode two sHsps each. Reporter gene studies suggested RNAT-mediated post-transcriptional regulation of shsp expression in both organisms. Detailed structural analysis of the two A. variabilis candidates revealed two novel RNAT types. The first, avashort, regulates translation primarily by masking of the AUG translational start codon. The second, featuring an extended initial hairpin, thus named avalong, presumably makes use of complex tertiary interaction. The 5′-UTR of the small heat shock gene hspA in the thermophile Thermosynechococcus elongatus is predicted to adopt an extended secondary structure. Structure probing revealed that the ribosome binding site was blocked at temperatures below 55 °C. The results of this study demonstrate that cyanobacteria commonly use RNATs to control expression of their small heat shock genes.

Transcriptional and Posttranscriptional Events Control Copper-Responsive Expression of a Rhodobacter capsulatus Multicopper Oxidase

The copper-regulated Rhodobacter capsulatus cutO (multicopper oxidase) gene confers copper tolerance and is carried in the tricistronic orf635-cutO-cutR operon. Transcription of cutO strictly depends on the promoter upstream of orf635, as demonstrated by lacZ reporter fusions to nested promoter fragments. Remarkably, orf635 expression was not affected by copper availability, whereas cutO and cutR were expressed only in the presence of copper. Differential regulation was abolished by site-directed mutations within the orf635-cutO intergenic region, suggesting that this region encodes a copper-responsive mRNA element. Bioinformatic predictions and RNA structure probing experiments revealed an intergenic stem-loop structure as the candidate mRNA element. This is the first posttranscriptional copper response mechanism reported in bacteria.

One gene, two proteins: Coordinated production of a copper chaperone by differential transcript formation and translational frameshifting in Escherichia coli

Programmed ribosomal frameshifting (PRF) is a translational anomaly causing the ribosome to shift into an alternative reading frame. PRFs are common in viral genomes, using a single nucleotide sequence to code for two proteins in overlapping frames. In bacteria and eukaryota, PRFs are less frequent. We report on a PRF in the copper detoxification system of Escherichia coli where a metallochaperone is generated out of the first 69 amino acids and a C‐terminal out‐of‐frame glycine of the gene copA. copA besides codes for the P1B‐ATPase CopA, a membrane‐integral protein and principal interaction target of the chaperone. To enhance the production of the frameshift‐generated cytosolic copper binding protein a truncated transcript is produced from the monocistronic copA gene. This shorter transcript is essential for producing sufficient amounts of the chaperone to support the membrane pump. The findings close the gap in our understanding of the molecular physiology of cytoplasmic copper transport in E. coli, revealing that a chaperone‐like entity is required for full functionality of the P1B‐ATPase copper pump. We, moreover, demonstrate that the primary transcriptional response to copper results in formation of the small transcript and concurrently, the metallochaperone plays a key role in resistance against copper shock.