Final Report Summary - HFQFOLD (STRUCTURAL BASIS OF SRNA-MEDIATED TRANSPOSITION REGULATION IN BACTERIA)
Main results and conclusions
In bacteria small RNAs (sRNAs) silence or activate target genes through base pairing with the mRNA and thereby modulating its translation. A central player in this process is the highly conserved RNA-chaperone Hfq, which facilitates the annealing of various sRNAs with their respective target mRNAs. In this project we aimed to understand Hfq mediated changes in s/mRNA structure and explain how it facilitates RNA annealing on an atomic level. Thus, our main goal was to characterize the structures of free and Hfq-bound s/mRNAs, as well as of a ternary Hfq:s/mRNA complex.
We succeeded solving a crystal structure of an Hfq:poly(A)-RNA complex, which provides novel insight into the mechanism of Hfq. For the first time, this structure visualizes an Hfq-mediated RNA-RNA interaction resembling a ternary Hfq:s/mRNA complex and will be of great importance to the community. The observed interaction is of a novel kind that could not be predicted and was thus never implicated in contributing to Hfq’s function. Several biophysical assays (analytical ultracentrifugation, electrophoretic mobility shift assays, isothermal titration calorimetry, fluorescence anisotropy) confirmed the structural arrangement in solution and its relevance for physiological RNA sequences. In addition, extensive computational analyses were conducted, that identified several further examples of the same interaction in diverse sRNA - mRNA pairs. This work also assigned sRNA structural elements to specific functions in the annealing process and showed that the affected sequences are conserved across various bacteria suggesting a general relevance for the novel mechanistic insights.
In addition to its role in RNA-RNA matchmaking, Hfq is implicated in contributing to RNA transcription and degradation via direct interaction with several proteins involved in these processes. To test this idea, we investigated Hfq’s interactions with core components of these machineries, the poly(A) polymerase I (PAP-1), RNAseE and transcription factor Rho in the presence or absence of s/mRNA. We characterized all interactions biochemically and mapped the minimal protein and RNA constructs required. While the interactions of Hfq with PAP-1 and RNAseE were strong and RNA mediated, Rho did not interact with Hfq in our assays.
In summary, our data provide fully unexpected insights into Hfq-mediated RNA annealing and display a novel way of RNA-mediated interaction in general. Biophysical and computational work clearly shows the physiological relevance of the data and suggests that the results are generally relevant to various bacteria. We believe that our results will substantially change the way in which the community will approach Hfq-mediated RNA annealing and they display a major advancement in the field of RNA-mediated regulation of gene expression.
Target groups
Our results will be of great interest to researchers in the field of small RNA regulation of gene expression, as well as structural an molecular biologists interested in protein RNA interactions and the functions of RNA in biology in general.
Photographs / project logo
None.
Project website
None
In bacteria small RNAs (sRNAs) silence or activate target genes through base pairing with the mRNA and thereby modulating its translation. A central player in this process is the highly conserved RNA-chaperone Hfq, which facilitates the annealing of various sRNAs with their respective target mRNAs. In this project we aimed to understand Hfq mediated changes in s/mRNA structure and explain how it facilitates RNA annealing on an atomic level. Thus, our main goal was to characterize the structures of free and Hfq-bound s/mRNAs, as well as of a ternary Hfq:s/mRNA complex.
We succeeded solving a crystal structure of an Hfq:poly(A)-RNA complex, which provides novel insight into the mechanism of Hfq. For the first time, this structure visualizes an Hfq-mediated RNA-RNA interaction resembling a ternary Hfq:s/mRNA complex and will be of great importance to the community. The observed interaction is of a novel kind that could not be predicted and was thus never implicated in contributing to Hfq’s function. Several biophysical assays (analytical ultracentrifugation, electrophoretic mobility shift assays, isothermal titration calorimetry, fluorescence anisotropy) confirmed the structural arrangement in solution and its relevance for physiological RNA sequences. In addition, extensive computational analyses were conducted, that identified several further examples of the same interaction in diverse sRNA - mRNA pairs. This work also assigned sRNA structural elements to specific functions in the annealing process and showed that the affected sequences are conserved across various bacteria suggesting a general relevance for the novel mechanistic insights.
In addition to its role in RNA-RNA matchmaking, Hfq is implicated in contributing to RNA transcription and degradation via direct interaction with several proteins involved in these processes. To test this idea, we investigated Hfq’s interactions with core components of these machineries, the poly(A) polymerase I (PAP-1), RNAseE and transcription factor Rho in the presence or absence of s/mRNA. We characterized all interactions biochemically and mapped the minimal protein and RNA constructs required. While the interactions of Hfq with PAP-1 and RNAseE were strong and RNA mediated, Rho did not interact with Hfq in our assays.
In summary, our data provide fully unexpected insights into Hfq-mediated RNA annealing and display a novel way of RNA-mediated interaction in general. Biophysical and computational work clearly shows the physiological relevance of the data and suggests that the results are generally relevant to various bacteria. We believe that our results will substantially change the way in which the community will approach Hfq-mediated RNA annealing and they display a major advancement in the field of RNA-mediated regulation of gene expression.
Target groups
Our results will be of great interest to researchers in the field of small RNA regulation of gene expression, as well as structural an molecular biologists interested in protein RNA interactions and the functions of RNA in biology in general.
Photographs / project logo
None.
Project website
None