SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the Metrology beamline.

Synchrotron X-ray footprinting complements the techniques commonly used to define the structure of molecules such as crystallography, small-angle X-ray scattering and nuclear magnetic resonance. It is remarkably useful in probing the structure and interactions of proteins with lipids, nucleic acids or with other proteins in solution, often better reflecting the in vivo state dynamics. To date, most X-ray footprinting studies have been carried out at the National Synchrotron Light Source, USA, and at the European Synchrotron Radiation Facility in Grenoble, France. This work presents X-ray footprinting of biomolecules performed for the first time at the X-ray Metrology beamline at the SOLEIL synchrotron radiation source. The installation at this beamline of a stopped-flow apparatus for sample delivery, an irradiation capillary and an automatic sample collector enabled the X-ray footprinting study of the structure of the soluble protein factor H (FH) from the human complement system as well as of the lipid-associated hydrophobic protein S3 oleosin from plant seed. Mass spectrometry analysis showed that the structural integrity of both proteins was not affected by the short exposition to the oxygen radicals produced during the irradiation. Irradiated molecules were subsequently analysed using high-resolution mass spectrometry to identify and locate oxidized amino acids. Moreover, the analyses of FH in its free state and in complex with complement C3b protein have allowed us to create a map of reactive solvent-exposed residues on the surface of FH and to observe the changes in oxidation of FH residues upon C3b binding. Studies of the solvent accessibility of the S3 oleosin show that X-ray footprinting offers also a unique approach to studying the structure of proteins embedded within membranes or lipid bodies. All the biomolecular applications reported herein demonstrate that the Metrology beamline at SOLEIL can be successfully used for synchrotron X-ray footprinting of biomolecules.

Characteristics of σ-dependent pausing by RNA polymerases from Escherichia coli and Thermus aquaticus.

The σ(70) subunit of RNA polymerase (RNAP) is the major transcription initiation factor in Escherichia coli. During transcription initiation, conserved region 2 of the σ(70) subunit interacts with the -10 promoter element and plays a key role in DNA melting around the starting point of transcription. During transcription elongation, the σ(70) subunit can induce pauses in RNA synthesis owing to interactions of region 2 with DNA regions similar to the -10 promoter element. We demonstrated that the major σ subunit from Thermus aquaticus (σ(A)) is also able to induce transcription pausing by T. aquaticus RNAP. However, hybrid RNAP containing the σ(A) subunit and E. coli core RNAP is unable to form pauses during elongation, while being able to recognize promoters and initiate transcription. Inability of the σ(A) subunit to induce pausing by E. coli RNAP is explained by the substitutions of non-conserved amino acids in region 2, in the subregions interacting with the RNAP core enzyme. Thus, changes in the structure of region 2 of the σ(70) subunit have stronger effects on transcription pausing than on promoter recognition, likely by weakening the interactions of the σ subunit with the core RNAP during transcription elongation.

Topography of lacUV5 initiation complexes.

Formation of a transcriptionally competent open complex is a highly regulated multistep process involving at least two intermediates. The rate of formation and stability of the intermediate complexes often determine promoter strength. However, the detailed mechanism of formation of the open complex and the high resolution structures of these intermediates are not known. In this study the structures of the open and intermediate complexes formed on the lacUV5 promoter by Escherichia coli RNA polymerase were analyzed using 'zero length' DNA-protein cross-linking. In both the open and the intermediate complexes the core subunits (ss' and ss) interact with lacUV5 DNA in a similar way, forming DNA-protein contacts flanking the initiation site. At the same time, the recognition (sigma(70)) subunit closely interacts with the promoter only in the open complex. In combination with our previous results, the data suggest that during promoter recognition contacts of the sigma subunit with core RNA polymerase and promoter DNA are rearranged in concert. These rearrangements constitute a landmark of transition from the intermediate to the open complex, identifying the sigma subunit as a key player directing formation of the open complex.

Differential melting of the transcription start site associated with changes in RNA polymerase-promoter contacts in initiating transcription complexes.

Formaldehyde cross-linking was used in a kinetic analysis of RNA polymerase-lacUV5 promoter interactions in open complexes (RP(o)). RP(o) quenched from 37 degrees C to 14 degrees C isomerised to a closed, competitor resistant, complex (RP(LT)). We observed that contacts of the beta' and sigma subunits with the positions -3, -5 of the non-template DNA strand disappeared very quickly during the first 30 seconds after the temperature downshift. The re-annealing of the DNA downstream of the transcription start site takes place in the same time scale. However re-annealing of the upstream part of the transcription bubble was slower and completed within five minutes. The results support a two-step model of promoter melting and suggest that conformational changes in the RNA polymerase occur concurrently with the melting around the transcription start site.

[Study of the structure of Escherichia coli RNA polymerase and its complex with the lacUV5-promotor using protein-protein and DNA-protein crosslinks, formed by formaldehyde]. / Issledovanie struktury RNK-polimerazy Escherichia coli i ee komplekxa s lacUV5-promotorom s pomoshch'iu belok-belkovykh i DNK-belkovykh sshivok, obrazuemykh formal'degidom.

The protein-protein and DNA-protein crosslinks produced by formaldehyde were used to investigate the intersubunit and subunit-DNA interactions for free RNA polymerase and for an open complex of RNA polymerase with the lacUV5 promoter. In both cases the contacts between beta,beta' and beta', sigma subunits were observed, while there were no contacts between beta and sigma subunits. Only one of beta or beta' subunits and a sigma subunit crosslink to promoter DNA. We have chosen the conditions for fixing the RNA polymerase-DNA complexes on different stages of transcription initiation. The possibility to use limited fixation with low concentrations of formaldehyde to study specific DNA-protein interactions was shown.

[Chromosomal proteins in chick embryo erythrocytes on transcriptionally active and inactive genes]. / Khromosomnye belki éritrotsitov émbrionov kur na transkriptsionno aktivnykh i neaktivnykh genakh.

Using chicken embryonic erythrocytes as a model, an experimental scheme for comparing the density of linker histones and high mobility group proteins on single-copy sequences of eukaryotic genome has been developed, thus permitting to probe alterations in the chromosomal protein pattern of transcribing chromatin. The report provides experimental evidence for validity of intracellular DNA-protein cross-linking, immunoaffinity chromatography and hybridization with single-stranded probes. Depletion of linker histones and enrichment of HMG 14/17 were shown to be the discriminating feature for transcriptionally active globin gene chromatin as opposed to inactive ovalbumin and lysozyme gene chromatin.

[Mutations in beta'-subunit of the Escherichia coli RNA-polymerase influence interaction with downstream duplex DNA in the elongation complex]. / Mutatsii v beta'-sub''edinitse RNK-polimerazy Escherichia coli, vliiaiushchie na vzaimodeistvie s perednim dupleksom DNK v élongatsionnom komplekse.

RNA polymerase (RNAP) exhibits absolute processivity being capable of synthesizing RNA 10(3)-10(5) nucleotides in length without breaking contact with the DNA template. Stability of the elongation complex is thought to depend, in particular, on the RNAP-DNA interactions downstream along the run of transcription. We studied the effects of several deletions and insertions in the RNAP beta'-subunit N-terminal region, which presumably interacts with the downstream duplex DNA in the elongation complex. Most of the mutations obtained led to gross defects in RNAP assembly and disturbed catalytic activity of the enzyme. The mutations reduced stability of both promoter and elongation complexes, probably because they altered the contacts between RNAP and the downstream duplex DNA.

Differences in contacts of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter are determined by core-enzyme of RNA polymerase.

The interaction of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter was studied at various temperatures. Using DNA-protein cross-linking induced by formaldehyde, it was demonstrated that each RNA polymerase formed a unique pattern of contacts with DNA in the open promoter complex. In the case of E. coli RNA polymerase, beta and sigma subunits were involved into formation of cross-links with the promoter, whereas in the case of T. aquaticus RNA polymerase its beta subunit formed the cross-links with the promoter. A cross-linking pattern in promoter complexes of a hybrid holoenzyme comprised of the core-enzyme of E. coli and sigma subunit of T. aquaticus was similar to that of the E. coli holoenzyme. This suggests that DNA-protein contacts in the promoter complex are primarily determined by the core-enzyme of RNA polymerase. However, temperature-dependent behavior of contact formation is determined by the sigma subunit. Results of the present study indicate that the method of formaldehyde cross-linking can be employed for elucidation of differences in the structure of promoter complexes of RNA polymerases from various bacteria.

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.

Identification of RNA polymerase beta' subunit segment contacting the melted region of the lacUV5 promoter.

Identification of the RNA polymerase functional regions involved in interactions with promoter is a basis for understanding the mechanism of transcription initiation. We have used formaldehyde cross-linking to identify a region of Escherichia coli RNA polymerase beta' subunit contacting lacUV5 promoter in open complex. Treatment of open complex with formaldehyde results in cross-linking of beta' and sigma(70) subunits at positions -5 and -3 on the nontemplate strand of the promoter DNA. These cross-links reflect specific interactions between RNA polymerase and promoter established in open complex. The positions of formaldehyde cross-links in the beta' subunit were mapped to the N-terminal segment (Cys(198)-Met(237)), which is contiguous to the evolutionary conserved region B. The proximity of the beta' and sigma cross-links suggest that the N-terminal region of the beta' subunit, interacting with single-stranded promoter DNA, can cooperate with the sigma subunit in the process of open complex formation.

Distribution of high mobility group proteins 1/2, E and 14/17 and linker histones H1 and H5 on transcribed and non-transcribed regions of chicken erythrocyte chromatin.

Quantitative analysis of distribution of chromosomal proteins on single copy DNA sequences has been further developed. Our approach consists of DNA-protein crosslinking within whole cells or isolated nuclei, specific immunoaffinity isolation of crosslinked complexes via protein and identification of crosslinked DNA by hybridisation with single-stranded DNA probes. The present study shows that transcribed chromatin of chicken embryonic erythrocyte beta globin gene is characterized by about 1.5-2.5-fold higher density of HMG 14/17 and 2-fold lower density of H1 and H5 as compared with non-transcribed chromatin of ovalbumin and lysozyme genes, whereas HMG 1/2, E proteins were equally distributed between DNA of both transcribed and non-transcribed genes. The depletion of H1/H5 in beta globin sequences was verified by the 'protein image' hybridisation technique (1). The DNase I hypersensitive site located 5' upstream from beta globin gene is deficient in all the proteins assayed, what implies a drastic disruption in the nucleosomal array. Minor quantitative changes of protein pattern suggest transient local perturbation of the chromatin on transcription.