Biomedical subjects

Marie-France Langelier

Publications and source records attributed to Marie-France Langelier.

6 recordsLinked to original sources

PubMed2006

Structural perspective on mutations affecting the function of multisubunit RNA polymerases.

High-resolution crystallographic structures of multisubunit RNA polymerases (RNAPs) have increased our understanding of transcriptional mechanisms. Based on a thorough review of the literature, we have compiled the mutations affecting the function of multisubunit RNA polymerases, many of which having been generated and studied prior to the publication of the first high-resolution structure, and highlighted the positions of the altered amino acids in the structures of both the prokaryotic and eukaryotic enzymes. The observations support many previous hypotheses on the transcriptional process, including the implication of the bridge helix and the trigger loop in the processivity of RNAP, the importance of contacts between the RNAP jaw-lobe module and the downstream DNA in the establishment of a transcription bubble and selection of the transcription start site, the destabilizing effects of ppGpp on the open promoter complex, and the link between RNAP processivity and termination. This study also revealed novel, remarkable features of the RNA polymerase catalytic mechanisms that will require additional investigation, including the putative roles of fork loop 2 in the establishment of a transcription bubble, the trigger loop in start site selection, and the uncharacterized funnel domain in RNAP processivity.

Amino Acid Substitution↗

PubMed2005

The highly conserved glutamic acid 791 of Rpb2 is involved in the binding of NTP and Mg(B) in the active center of human RNA polymerase II.

During transcription by RNA polymerase (RNAP) II, the incoming ribonucleoside triphosphate (NTP) enters the catalytic center in association with an Mg2+ ion, termed metal B [Mg(B)]. When bound to RNAP II, Mg(B) is coordinated by the beta and gamma phosphates of the NTP, Rpb1 residues D481 and D483 and Rpb2 residue D837. Rpb2 residue D837 is highly conserved across species. Notably, its neighboring residue, E836 (E791 in human RNAP II), is also highly conserved. To probe the role of E791 in transcription, we have affinity purified and characterized a human RNAP II mutant in which this residue was substituted for alanine. Our results indicate that the transcription activity of the Rpb2 E791A mutant is impaired at low NTP concentrations both in vitro and in vivo. They also revealed that both its NTP polymerization and transcript cleavage activities are decreased at low Mg concentrations. Because Rpb2 residue E791 appears to be located too far from the NTP-Mg(B) complex to make direct contact at either the entry (E) or addition (A) site, we propose alternative mechanisms by which this highly conserved residue participates in loading NTP-Mg(B) in the active site during transcription.

Amino Acid Substitution↗

PubMed2005

Functional dissection of the catalytic mechanism of mammalian RNA polymerase II.

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.

Animals↗

PubMed2004

Interaction networks of the molecular machines that decode, replicate, and maintain the integrity of the human genome.

The interaction of many proteins with genomic DNA is required for the expression, replication, and maintenance of the integrity of mammalian genomes. These proteins participate in processes as diverse as gene transcription and mRNA processing, as well as in DNA replication, recombination, and repair. This intricate system, where the various nuclear machineries interact with one another and bind to either common or distinct DNA regions to create an impressive network of protein-protein and protein-DNA interactions, is made even more complex by the need for a very stringent control in order to ensure normal cell growth and differentiation. A general methodology based on the in vivo pull-down of tagged components of nuclear machines and regulatory proteins was used to study genome-wide protein-protein and protein-DNA interactions in mammalian cells. In particular, this approach has been used in defining the interaction networks (or "interactome") formed by RNA polymerase II, a molecular machine that decodes the human genome. In addition, because this methodology allows for the purification of variant forms of tagged complexes having site-directed mutations in key elements, it can also be used for deciphering the relationship between the structure and the function of the molecular machines, such as RNA polymerase II, that by binding DNA play a central role in the pathway from the genome to the organism.

DNA↗

PubMed2004

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

Animals↗

PubMed2004

Photo-cross-linking of a purified preinitiation complex reveals central roles for the RNA polymerase II mobile clamp and TFIIE in initiation mechanisms.

The topological organization of a TATA binding protein-TFIIB-TFIIF-RNA polymerase II (RNAP II)-TFIIE-promoter complex was analyzed using site-specific protein-DNA photo-cross-linking of gel-purified complexes. The cross-linking results for the subunits of RNAP II were used to determine the path of promoter DNA against the structure of the enzyme. The results indicate that promoter DNA wraps around the mobile clamp of RNAP II. Cross-linking of TFIIF and TFIIE both upstream of the TATA element and downstream of the transcription start site suggests that both factors associate with the RNAP II mobile clamp. TFIIE alpha closely approaches promoter DNA at nucleotide -10, a position immediately upstream of the transcription bubble in the open complex. Increased stimulation of transcription initiation by TFIIE alpha is obtained when the DNA template is artificially premelted in the -11/-1 region, suggesting that TFIIE alpha facilitates open complex formation, possibly through its interaction with the upstream end of the partially opened transcription bubble. These results support the central roles of the mobile clamp of RNAP II and TFIIE in transcription initiation.

Animals↗