Final Report Summary - HSRRNA (Structural and functional studies of structured RNA involved in heat shock response)
Specimens of bacterial and eukaryotic non-coding RNA were prepared in large scale and in high purity. The genes encoding the regulatory RNAs were cloned into the pUC19 vector and used as template in PCR reactions to prepare templates with a T7 promoter for in vitro transcription. Samples were purified to homogeneity and protocols developed for optimal sample renaturation and preparation of conformational homogeneous specimens suitable for crystallisation experiments. Extensive crystallisation surveys were undertaken of the RNA and its complexes with protein partners using a nanoliter dispensing robot and manually with custom designed screens. Small crystals were obtained and screened at the Diamond Light Source, but none yielded diffraction data. The solution structure of the 6S non-coding RNA was characterised with biophysical methods. Secondary structure was investigated with in-line probing experiments and its stability characterised by thermal melting assays and fluorescence-based thermal shift assays. Changes of the hydrodynamic radius as a function of temperature where recording by Dynamic Light Scattering (DLS), while the sedimentation coefficient, estimation of the shape of the molecule and conformational changes related with the ionic strength were performed by Analytical Ultracentrifugation (AUC) experiments. Structural features of the tertiary structure were revealed by far-UV Circular Dichroism measurements (CD).
The complex formation of this RNA with protein partners was also characterized using biophysical and spectroscopic techniques. We first verified that the methods used for production and purification of this RNA did not interfere with its native folding and activity. The complex formation of the 6S non-coding RNA with its native protein partner, the RNA polymerase, was demonstrated by nanoflow electrospray mass spectrometry, which also revealed the stoichiometry of this complex. These data identified the predicted molecular mass for the RNA, showing that it did not contain any modifications, and suggesting that non-coding RNA function does not require covalent modification for activity. Interaction of the 6S RNA with RNase E, which is the ribonuclease which performs its maturation, were evaluated using mobility shift analysis, gel filtration, analytical ultracentrifugation and isothermal titration calorimetry experiments. The process of maturation of this RNA by ribonuclease RNase E was also studied by biochemical assays. For this purpose time course experiments and fixed-time biochemical assays were performed to estimate the kinetics of the processing reaction that converts the precursor RNA to its mature form.
Our studies demonstrate that 6S RNA folds into an elongated molecule with highly stable secondary structure and much weaker tertiary interactions. The overall structure seems to be dominated by an A-type helix. Earlier studies of the interaction of 6S with s70-RNAP holoenzyme have evaluated the site of interaction. In this study, we have been able to observe the ternary complex using nanoflow ESI mass spectrometry, which establishes the molecular stoichiometry of the assembly and shows that a single molecule of 6S binds a single holoenzyme assembly, without loss of any of the canonical components of the holoenzyme.
The precursor form of this RNA is efficiently processed by RNase E and generates a stable mature form that is not further digested by the endoribonuclease. In contrast, the fragments remaining from the precursor appear to be susceptible to continued degradation. Our experiments indicate that the enzyme activity is inhibited at high concentrations of substrate. The kinetics of the burst phase suggest that it may be either the substrate, the product, or both that repress the activity of RNase E.
RNase E forms the scaffold of the multi-enzyme RNA degradosome, which recruits additional RNA binding components into a large energy dependent machine. We observed that a reconstituted degradosome assembly that has a catalytically inactive mutant of RNase E can form a stable complex with 6S that persists in agarose gels. The stability is much greater than for the isolated catalytic domain, suggesting that the additional binding sites for RNA influence the association with the substrate. However, preliminary processing experiments of precursor 6S by active, endogenous-purified full degradosome indicated a similar degree of inhibition as that observed with the catalytic domain of RNase E. These findings imply that the inhibition may have a biological role in the processing of 6S RNA.
Extension of the project to a direction, which concerns investigation of the interactions of this RNA with the RNA polymerase and structural characterisation of the complex, has been favourably evaluated by independent experts and has been invited for negotiation in the framework of a Marie Curie FP7-PEOPLE-2010 Reintegration Grant.
Our research has shed light on a very new field of the biological sciences that of the non-coding RNAs which only recently have been identified as major modulators of gene expression at various cellular levels in all kingdoms of life. Their mechanism of action is extremely diverse which indicates that so far we have only had a glimpse on this new regulatory factor. The RNA under study is a transcriptional regulator and thus our research has have bearing on understanding the mechanism of the highly conserved and functionally important transcriptional response. The transcriptional mechanism is a proven target for broad-spectrum antibacterial therapy. Although the research is still in a preliminary stage, the information obtained may be eventually useful for chemical intervention of the transcriptional regulation either as a research tool to investigate the response itself, or as lead reagents for drug design.
The complex formation of this RNA with protein partners was also characterized using biophysical and spectroscopic techniques. We first verified that the methods used for production and purification of this RNA did not interfere with its native folding and activity. The complex formation of the 6S non-coding RNA with its native protein partner, the RNA polymerase, was demonstrated by nanoflow electrospray mass spectrometry, which also revealed the stoichiometry of this complex. These data identified the predicted molecular mass for the RNA, showing that it did not contain any modifications, and suggesting that non-coding RNA function does not require covalent modification for activity. Interaction of the 6S RNA with RNase E, which is the ribonuclease which performs its maturation, were evaluated using mobility shift analysis, gel filtration, analytical ultracentrifugation and isothermal titration calorimetry experiments. The process of maturation of this RNA by ribonuclease RNase E was also studied by biochemical assays. For this purpose time course experiments and fixed-time biochemical assays were performed to estimate the kinetics of the processing reaction that converts the precursor RNA to its mature form.
Our studies demonstrate that 6S RNA folds into an elongated molecule with highly stable secondary structure and much weaker tertiary interactions. The overall structure seems to be dominated by an A-type helix. Earlier studies of the interaction of 6S with s70-RNAP holoenzyme have evaluated the site of interaction. In this study, we have been able to observe the ternary complex using nanoflow ESI mass spectrometry, which establishes the molecular stoichiometry of the assembly and shows that a single molecule of 6S binds a single holoenzyme assembly, without loss of any of the canonical components of the holoenzyme.
The precursor form of this RNA is efficiently processed by RNase E and generates a stable mature form that is not further digested by the endoribonuclease. In contrast, the fragments remaining from the precursor appear to be susceptible to continued degradation. Our experiments indicate that the enzyme activity is inhibited at high concentrations of substrate. The kinetics of the burst phase suggest that it may be either the substrate, the product, or both that repress the activity of RNase E.
RNase E forms the scaffold of the multi-enzyme RNA degradosome, which recruits additional RNA binding components into a large energy dependent machine. We observed that a reconstituted degradosome assembly that has a catalytically inactive mutant of RNase E can form a stable complex with 6S that persists in agarose gels. The stability is much greater than for the isolated catalytic domain, suggesting that the additional binding sites for RNA influence the association with the substrate. However, preliminary processing experiments of precursor 6S by active, endogenous-purified full degradosome indicated a similar degree of inhibition as that observed with the catalytic domain of RNase E. These findings imply that the inhibition may have a biological role in the processing of 6S RNA.
Extension of the project to a direction, which concerns investigation of the interactions of this RNA with the RNA polymerase and structural characterisation of the complex, has been favourably evaluated by independent experts and has been invited for negotiation in the framework of a Marie Curie FP7-PEOPLE-2010 Reintegration Grant.
Our research has shed light on a very new field of the biological sciences that of the non-coding RNAs which only recently have been identified as major modulators of gene expression at various cellular levels in all kingdoms of life. Their mechanism of action is extremely diverse which indicates that so far we have only had a glimpse on this new regulatory factor. The RNA under study is a transcriptional regulator and thus our research has have bearing on understanding the mechanism of the highly conserved and functionally important transcriptional response. The transcriptional mechanism is a proven target for broad-spectrum antibacterial therapy. Although the research is still in a preliminary stage, the information obtained may be eventually useful for chemical intervention of the transcriptional regulation either as a research tool to investigate the response itself, or as lead reagents for drug design.