Regulation of promoter activity through electrostatic interactions with RNA polymerase

Electrostatic potential profiles were calculated for σ⁷⁰-specific phage T4 promoters and four consensus-like synthetic promoters. Promoters could be classified according to the presence of specific elements of the electrostatic profile in the farther upstream region (?60 to ?100). A correlation was found between the kind of such specific element and the functional behavior of the promoter. A general scheme is proposed whereby the promoter activity is regulated through electrostatic interactions between the upstream region of promoter DNA and the RNA polymerase α-subunit.     

Conformational changes in E. coli RNA polymerase during promoter recognition.

We analysed complexes formed during recognition of the lacUV5 promoter by E. coli RNA polymerase using formaldehyde as a DNA-protein and protein-protein cross-linking reagent. Most of the cross-linked complexes specific for the open complex (RPO) contain the beta' subunit of RNA polymerase cross-linked with promoter DNA in the regions: -50 to -49; -5 to -10; + 5 to +8 and +18 to +21. The protein-protein cross-linking pattern of contacting subunits is the same for the RNA polymerase in solution and in RPO: there are strong sigma-beta' and beta-beta' interactions. In contrast, only beta-beta' cross-links were detected in the closed (RPC) and intermediate (RPI) complexes. In presence of lac repressor before or after formation of the RPO cross-linking pattern is similar with that of RPI (RPC) complex.     

RNA profiling in host-pathogen interactions

The development of novel anti-bacterial treatment strategies will be aided by an increased understanding of the interactions that take place between bacteria and host cells during infection. Global expression profiling using microarray technologies can help to describe and define the mechanisms required by bacterial pathogens to cause disease and the host responses required to defeat bacterial infection.     

Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses

Permeabilization of the Endoplasmic Reticulum (ER) is instrumental in the progression of host-cell infection by many viral pathogens. We have described that permeabilization of ER model membranes by the pore-forming domain of the Classical Swine Fever Virus (CSFV) p7 protein depends on two sequence determinants: the C-terminal transmembrane helix, and the preceding polar loop that regulates its activity. Here, by combining ion-channel activity measurements in planar lipid bilayers with imaging of single Giant Unilamellar Vesicles (GUVs), we demonstrate that point substitutions directed to conserved residues within these regions affect ER-like membrane permeabilization following distinct mechanisms. Whereas the polar loop appeared to be involved in protein insertion and oligomerization, substitution of residues predicted to face the lumen of the pore inhibited large conducting channels (>1 nS) over smaller ones (120 pS). Quantitative analyses of the ER-GUV distribution as a function of the solute size revealed a selective inhibition for the permeation of solutes with sizes larger than 4 kDa, further demonstrating that the mutation targeting the transmembrane helix prevented formation of the large pores. Collectively, our data support the idea that the pore-forming domain of p7 may assemble into finite pores with approximate diameters of 1 and 5 nm. Moreover, the observation that the mutation interfering with formation of the larger pores can hamper virus production without affecting ER localization or homo-oligomerization, suggests prospective strategies to block/attenuate pestiviruses.     

Transient RNA-protein interactions in RNA folding

The RNA folding trajectory features numerous off-pathway folding traps, which represent conformations that are often equally as stable as the native functional ones. Therefore, the conversion between these off-pathway structures and the native correctly folded ones is the critical step in RNA folding. This process, referred to as RNA refolding, is slow, and is represented by a transition state that has a characteristic high free energy. Because this kinetically limiting process occurs in vivo, proteins (called RNA chaperones) have evolved that facilitate the (re)folding of RNA molecules. Here, we present an overview of how proteins interact with RNA molecules in order to achieve properly folded states. In this respect, the discrimination between static and transient interactions is crucial, as different proteins have evolved a multitude of mechanisms for RNA remodeling. For RNA chaperones that act in a sequence-unspecific manner and without the use of external sources of energy, such as ATP, transient RNA-protein interactions represent the basis of the mode of action. By presenting stretches of positively charged amino acids that are positioned in defined spatial configurations, RNA chaperones enable the RNA backbone, via transient electrostatic interactions, to sample a wider conformational space that opens the route for efficient refolding reactions.     

Virus-host protein interactions in RNA viruses

RNA viruses exhibit small-sized genomes that only encode a limited number of viral proteins, but still establish complex networks of interactions with host cell components. Here we summarize recent reports that aim at understanding general features of RNA virus infection networks at the protein level.     

CAP and RNA polymerase interactions with the lac promoter: binding stoichiometry and long range effects.

The binding stoichiometries of the complexes formed when the E. coli cyclic AMP receptor protein (CAP) binds to 203 bp lac promoter-operator restriction fragments have been determined. Under quantitative binding conditions, a single dimer of CAP occupies each of two sites in the promoter. Different electrophoretic mobilities are observed for 1:1 complexes formed with L8-UV5 mutant, L305 mutant, and wild type promoter fragments, indicating sequence-specific structural differences between the complexes. The differences in gel mobility between L8-UV5 and wild type complexes disappear when the promoter fragments are cleaved with Hpa II restriction endonuclease. Models in which CAP alters DNA conformation or in which CAP forms a transient intramolecular bridge between two domains of a DNA molecule could account for these observations. The selective binding of RNA polymerase to CAP-promoter complexes is demonstrated: the binding of a single CAP dimer to the promoter is sufficient to stimulate subsequent polymerase binding. Functional CAP molecules are not released from the promoter on polymerase binding.     

RNA:RNA interactions in the large subunit ribosomal RNA of Euglena gracilis

In Euglena gracilis, the cytoplasmic large subunit (LSU) rRNA is composed of 14 discrete small RNA species that must somehow interact in the functional ribosome. We have isolated native complexes of Euglena rRNA and show here that the largest of these complexes contains eight of the 14 LSU rRNA species. Several of these small rRNA species are able to associate in vitro to reform an isolated domain of LSU rRNA structure.