Mathieu Rappas

Modus operandi of the bacterial RNA polymerase containing the σ54 promoter‐specificity factor

Bacterial sigma (σ) factors confer gene specificity upon the RNA polymerase, the central enzyme that catalyses gene transcription. The binding of the alternative σ factor σ54 confers upon the RNA polymerase special functional and regulatory properties, making it suited for control of several major adaptive responses. Here, we summarize our current understanding of the interactions the σ54 factor makes with the bacterial transcription machinery.

Regulation of DNA Replication through Sld3-Dpb11 Interaction Is Conserved from Yeast to Humans

Cyclin-dependent kinases (CDKs) play crucial roles in promoting DNA replication and preventing rereplication in eukaryotic cells [1-4]. In budding yeast, CDKs promote DNA replication by phosphorylating two proteins, Sld2 and Sld3, which generates binding sites for pairs of BRCT repeats (breast cancer gene 1 [BRCA1] C terminal repeats) in the Dpb11 protein [5, 6]. The Sld3-Dpb11-Sld2 complex generated by CDK phosphorylation is required for the assembly and activation of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase. In response to DNA replication stress, the interaction between Sld3 and Dpb11 is blocked by the checkpoint kinase Rad53 [7], which prevents late origin firing [7, 8]. Here we show that the two key CDK sites in Sld3 are conserved in the human Sld3-related protein Treslin/ticrr and are essential for DNA replication. Moreover, phosphorylation of these two sites mediates interaction with the orthologous pair of BRCT repeats in the human Dpb11 ortholog, TopBP1. Finally, we show that DNA replication stress prevents the interaction between Treslin/ticrr and TopBP1 via the Chk1 checkpoint kinase. Our results indicate that Treslin/ticrr is a genuine ortholog of Sld3 and that the Sld3-Dpb11 interaction has remained a critical nexus of S phase regulation through eukaryotic evolution.

Organization of an activator-bound RNA polymerase holoenzyme.

Summary Transcription initiation involves the conversion from closed promoter complexes, comprising RNA polymerase (RNAP) and double-stranded promoter DNA, to open complexes, in which the enzyme is able to access the DNA template in a single-stranded form. The complex between bacterial RNAP and its major variant sigma factor σ54 remains as a closed complex until ATP hydrolysis-dependent remodeling by activator proteins occurs. This remodeling facilitates DNA melting and allows the transition to the open complex. Here we present cryoelectron microscopy reconstructions of bacterial RNAP in complex with σ54 alone, and of RNAP-σ54 with an AAA+ activator. Together with photo-crosslinking data that establish the location of promoter DNA within the complexes, we explain why the RNAP-σ54 closed complex is unable to access the DNA template and propose how the structural changes induced by activator binding can initiate conformational changes that ultimately result in formation of the open complex.

Molecular Determinants for PspA-Mediated Repression of the AAA Transcriptional Activator PspF

The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the sigma(54) subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of sigma(54) AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own expression. The PspA protein is distributed between the cytoplasm and the inner membrane fraction. In addition to its transcriptional inhibitory role, PspA assists maintenance of the proton motive force and protein export. Several lines of in vitro evidence indicate that PspA-PspF interactions inhibit the ATPase activity of PspF, resulting in the inhibition of PspF-dependent gene expression. In this study, we characterize sequences within PspA and PspF crucial for the negative effect of PspA upon PspF. Using a protein fragmentation approach, we show that the integrity of the three putative N-terminal alpha-helical domains of PspA is crucial for the role of PspA as a negative regulator of PspF. A bacterial two-hybrid system allowed us to provide clear evidence for an interaction in E. coli between PspA and PspF in vivo, which strongly suggests that PspA-directed inhibition of PspF occurs via an inhibitory complex. Finally, we identify a single PspF residue that is a binding determinant for PspA.

Different Quaternary Structures of Human RECQ1 Are Associated with Its Dual Enzymatic Activity

RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPγS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 Å × 130 Å with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding.

Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1.

TopBP1 is a scaffold protein that coordinates activation of the DNA-damage-checkpoint response by coupling binding of the 9-1-1 checkpoint clamp at sites of ssDNA, to activation of the ATR-ATRIP checkpoint kinase complex. We have now determined the crystal structure of the N-terminal region of human TopBP1, revealing an unexpected triple-BRCT domain structure. The arrangement of the BRCT domains differs significantly from previously described tandem BRCT domain structures, and presents two distinct sites for binding phosphopeptides in the second and third BRCT domains. We show that the site in the second but not third BRCT domain in the N-terminus of TopBP1, provides specific interaction with a phosphorylated motif at pSer387 in Rad9, which can be generated by CK2.TopBP1 is a scaffold protein that coordinates activation of the DNA-damage-checkpoint response by coupling binding of the 9-1-1 checkpoint clamp at sites of ssDNA, to activation of the ATR–ATRIP checkpoint kinase complex. We have now determined the crystal structure of the N-terminal region of human TopBP1, revealing an unexpected triple-BRCT domain structure. The arrangement of the BRCT domains differs significantly from previously described tandem BRCT domain structures, and presents two distinct sites for binding phosphopeptides in the second and third BRCT domains. We show that the site in the second but not third BRCT domain in the N-terminus of TopBP1, provides specific interaction with a phosphorylated motif at pSer387 in Rad9, which can be generated by CK2.

Coupling nucleotide hydrolysis to transcription activation performance in a bacterial enhancer binding protein

The bacterial enhancer binding proteins (bEBP) are members of the AAA+ protein family and have a highly conserved ‘DE’ Walker B motif thought to be involved in the catalytic function of the protein with an active role in nucleotide hydrolysis. Based on detailed structural data, we analysed the functionality of the conserved ‘DE’ Walker B motif of a bEBP model, phage shock protein F (PspF), to investigate the role of these residues in the σ54‐dependent transcription activation process. We established their role in the regulation of PspF self‐association and in the relay of the ATPase activity to the remodelling of an RNA polymerase·promoter complex (Eσ54·DNA). Specific substitutions of the conserved glutamate (E) allowed the identification of new functional ATP·bEBP·Eσ54 complexes which are stable and transcriptionally competent, providing a new tool to study the initial events of the σ54‐dependent transcription activation process. In addition, we show the importance of this glutamate residue in σ54·DNA conformation sensing, permitting the identification of new intermediate stages within the transcription activation pathway.

Protein‐induced DNA bending clarifies the architectural organization of the σ54‐dependent glnAp2 promoter

σ54‐RNA polymerase (Eσ54) predominantly contacts one face of the DNA helix in the closed promoter complex, and interacts with the upstream enhancer‐bound activator via DNA looping. Up to date, the precise face of Eσ54 that contacts the activator to convert the closed complex to an open one remains unclear. By introducing protein‐induced DNA bends at precise locations between upstream enhancer sequences and the core promoter of the σ54‐dependent glnAp2 promoter without changing the distance in‐between, we observed a strong enhanced or decreased promoter activity, especially on linear DNA templates in vitro. The relative positioning and orientations of Eσ54, DNA bending protein and enhancer‐bound activator on linear DNA were determined by in vitro footprinting analysis. Intriguingly, the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix compared with the DNA bound Eσ54 in the closed complex. Therefore, these results provide evidence that the activator must approach the Eσ54 closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. This proposal is further supported by the modelling of activator‐promoter DNA‐Eσ54 complex.

The Second Paradigm for Activation of Transcription

Publisher Summary Gene transcription is central to the development, differentiation, and adaptation of cells. Control of transcription requires the interplay of signaling pathways with the molecular machinery of transcription, the DNA-dependent RNA polymerase (RNAP) enzyme, regulatory proteins that act upon it, and the nucleic acid that is transcribed. The genetic tractability of bacteria, in particular Escherichia coli and Bacillus subtilis, and yeast has allowed rapid progress in elucidating the types of strategy used for the control of gene expression at the level of transcription. The RNAP is evolutionarily conserved in sequence, structure, and function from bacteria to humans. The simple (in terms of subunit composition) bacterial RNAP is an excellent model system to study the control of gene transcription. This chapter also describes the components of such a system and how they interact to allow regulation of RNAP activity at the level of the DNA opening event (i.e., open complex formation) necessary for trancription initiation.

A second paradigm for gene activation in bacteria

Control of gene expression is key to development and adaptation. Using purified transcription components from bacteria, we employ structural and functional studies in an integrative manner to elaborate a detailed description of an obligatory step, the accessing of the DNA template, in gene expression. Our work focuses on a specialized molecular machinery that utilizes ATP hydrolysis to initiate DNA opening and permits a description of how the events triggered by ATP hydrolysis within a transcriptional activator can lead to DNA opening and transcription. The bacterial EBPs (enhancer binding proteins) that belong to the AAA(+) (ATPases associated with various cellular activities) protein family remodel the RNAP (RNA polymerase) holoenzyme containing the sigma(54) factor and convert the initial, transcriptionally silent promoter complex into a transcriptionally proficient open complex using transactions that reflect the use of ATP hydrolysis to establish different functional states of the EBP. A molecular switch within the model EBP we study [called PspF (phage shock protein F)] is evident, and functions to control the exposure of a solvent-accessible flexible loop that engages directly with the initial RNAP promoter complex. The sigma(54) factor then controls the conformational changes in the RNAP required to form the open promoter complex.