Post-transcriptional Regulation by small non-coding RNAs and Hfq in Eubacteria and by Initiation Factors in Archaea

 

Regulation of translation initiation in response to environmental stimuli has become a major theme in bacterial gene expression and pathogenicity.  Our main interests focus on translational control mechanisms exerted by small regulatory RNAs and by the RNA chaperone Hfq in Eubacteria as well as on the regulatory function of different initiation factors on translation in the archaeon Sulfolobus solfataricus.

 

Hfq, sRNAs and bacterial virulence. 

Bacteria are constantly challenged by changing environmental conditions.  They employ a number of translational control mechanisms including trans-acting proteins, small non-coding RNAs (sRNAs) as well as features inherent to mRNA structure, which permit a fast adaptation to new environments or to different kinds of stress.  E. coli host factor I (Hfq) has a hexameric ring-shaped structure and belongs to the large family of Sm and Sm-like proteins with RNA binding activity.   The work on Hfq was initiated by our observation that translation initiation of the E. coli ompA gene is mediated by Hfq.  This work directly demonstrated that a 30S ribosome bound to the ompA 5'-UTR protected the transcript from RNase E cleavage, and thereby contributed to a better understanding of the interrelation between translation (initiation) and mRNA turnover.   Subsequent work revealed that Hfq induces structural changes in RNA and that it functions as genuine RNA chaperone.  In Gram-negative Bacteria, Hfq acts as a global regulator involved in post-transcriptional regulation and virulence.  Hfq binds to a number of sRNAs, contributes to their stability, and is often required for translational silencing and activation mediated by these riboregulators.  At present, it is poorly understood how the protein interacts with sRNAs and their mRNA substrates.  Therefore, a collaborative effort between my group and the Djinovic-Carugo group (MFPL, Vienna Biocenter) concerns the elucidation of the complete 3D-structure of Hfq and that of Hfq-RNA complexes.  Hfq has been shown to localize to the 30S ribosomal subunit.  Ongoing research projects are aimed at defining (i) the molecular environment of Hfq on the 30S subunit, (ii) Hfq interacting proteins involved in post-transcriptional processes, (iii) the molecular events underlying sRNA-based mRNA silencing, and (iv) the molecular events leading to decay or stabilization of sRNA/mRNAs complexes.  In the context of (iii), we were recently able to demonstrate that a sRNA does not necessarily regulate translation of a target mRNA in a direct manner, i.e. by binding to or in the vicinity of its ribosome binding site but can do so by regulation of an upstream open reading frame to which the downstream gene is translationally coupled.  In addition, this work provided evidence for an iron-dependent decoding mechanism of mRNAd encoding iron-containing proteins.

Pseudomonas aeruginosa (PAO1) is a major cause of hospital acquired infections, exerts the highest case fatality rate among Gram-negative pathogens, and contributes to mortality in cystic fibrosis patients.  As we have previously shown that a PAO1 hfq- mutant was significantly attenuated in virulence, it can be anticipated that ncRNAs are involved in regulation of many virulence genes.  The complexity of post-transcriptional regulation mediated by Hfq became apparent when we compared the transcriptome profiles of PAO1 with the of an isogenic hfq- mutant.  The complex regulatory circuit affected by Hfq involved stabilization of a regulatory RNA, which indirectly affects a regulatory cascade, mediating virulence gene expression. The ongoing and future P. aeruginosa project, which is currently the major topic in my laboratory, is concentrating on the following topics: (i) detection of sRNAs under various stress conditions, including identification of Hfq-binding sRNAs from clinical P. aeruginosa strains present in the sputum of cystic fibrosis patients, and (ii) validation of their potential target genes using biochemical and genetic methods.  It is anticipated that these approaches will reveal sRNAs involved in virulence gene expression, i.e. in post-transcriptional regulation of the PAO1 pathogenome.  To this end we have obtained evidence that some of the identified and characterized sRNAs impact on multi-drug resistance in PAO1 and on synthesis of the virulence factor pyocyanin.

 

Post-transcriptional regulation in the archaeon Sulfolobus solfataricus. 

In contrast to Bacteria and Eukaryotes, the process of protein biosynthesis (translation), particularly that of translation initiation, is poorly understood in Archaea, representing the third domain of life.  We have chosen the thermophilic archaeon S. solfataricus as a model system because its genome sequence is completed.  This allowed cloning and purification of different translation initiation factors, the functions of which we are currently exploring.  We were able to demonstrate that the trimeric SSO translation initiation factor 2 (a/eIF2) is, like its eukaryotic counterpart eIF2, pivotal for delivery of initiator tRNA to the ribosome.  However, in contrast to the eukaryal protein, the γ-subunit of a/eIF2 does possesses an intrinsic capacity for tRNAi binding, which could suggest that the γ-subunit functioned as a monomer earlier in evolution.  Nevertheless, a stable interaction with charged tRNAi required the presence of both, the α- and γ-subunits, whereas in Eukaryotes the α- and β-subunit are required.  These differences can be explained in light of the crystal structures of the a/eIF2-γ subunit and that of the trimeric factor, which have been elucidated in collaboration with the Garber laboratory (Russian Academy of Science, Pushchino).  Moreover, we were able to show that the a/eIF2 γ-subunit exhibits an additional function with resemblance to the eukaryotic cap-complex.  It binds to the 5´-triphosphate end of mRNAs and counteracts 5´- to 3´- mRNA decay in SSO.  This unprecedented capacity of the archaeal initiation factor further indicates that 5´→ 3´ directional mRNA decay is a pathway common to all domains of life.  These intriguing observations provide a basis for future studies towards the function of the a/eIF2-γ subunit in SSO mRNA metabolism.  In contrast to energetically favorable chemoorganotrophic growth, Sulfolobus also thrives in sulfur-rich hot springs, where it grows chemolithotrophically.  In future research we will address questions concerning the protective role of a/eF2-γ in long-term survival under famine conditions, when the synthesis of ribosomes is likely to be decreased.  These studies are also expected to contribute to archaeal Molecular Biotechnology in that a better understanding of post-transcriptional processes in Archaea can benefit the use of these organisms for the production of valuable heat stable enzymes.

 

Phage-derived Proteins as novel Antimicrobials

The appearance of multi-resistant bacterial pathogens has become a serious threat.  An alternative to antibiotics to treat bacterial infections is the use of bacteriophage-derived proteins.  A biotechnology driven project focuses on engineering of genetically modified antimicrobial peptides specific for the human pathogen Staphylococcus aureus with the aim to generate safe and highly specific therapeutic agents.