Ann Hochschild

Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.

Cooperative binding of λ repressors to sites separated by integral turns of the DNA helix

Abstract λ repressors bind cooperatively to adjacent pairs of operator sites. Here we show that repressors bind cooperatively to pairs of operator sites whose centers have been separated by five or six turns of the helix. No cooperativity is observed when the centers of these sites are on opposite sides of the DNA helix. Cooperativity depends upon the same part of the protein (the carboxyl domain) that mediates cooperativity when the sites are adjacent. As the repressors bind, the DNA between the sites becomes alternately sensitive and resistant to DNAase I cleavage at half turn intervals. We suggest that when repressors bind cooperatively to separated sites, the DNA forms a loop, thus allowing the two repressors to touch.

Repressor structure and the mechanism of positive control

It has been suggested that the lambda repressor stimulates transcription of its own gene by binding to the lambda operator and contacting RNA polymerase bound to the adjacent promoter. We describe three different mutants (called pc) of the lambda phage repressor that are specifically deficient in the positive control function. We show that the amino acid residues altered in the pc mutants lie on the surface of the DNA-bound repressor that we predict, based on structural and other evidence, would most closely approach DNA-bound polymerase. Furthermore, we describe a pc mutant of the P22 repressor. We argue that in both the lambda and P22 repressors a structure comprised of two alpha helices has two functions: to bind DNA and to contact RNA polymerase. In the two cases, however, different regions of this structure contact polymerase to mediate positive control.

Structural Basis for Bacterial Transcription-Coupled DNA Repair

Coupling of transcription and DNA repair in bacteria is mediated by transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the transcription elongation complex, provide mechanistic insights into TRCF function.

Protein–Protein Contacts that Activate and Repress Prokaryotic Transcription

Recently the structures of two of the DNA-binding domains of RNAP, the α-CTD (7xJeon, Y.H, Negishi, T, Shirakawa, M, Yamazaki, T, Fujita, N, Ishihama, A, and Kyogoku, Y. Science. 1995; 270: 1495–1497Crossref | PubMedSee all References, 6xGaal, T, Ross, W, Blatter, E.E, Tang, H, Jia, X, Krishnan, V.V, Assa-Munt, N, Ebright, R.H, and Gourse, R.L. Genes Dev. 1996; 10: 16–26Crossref | PubMedSee all References), and a portion of σ70 containing the −10 region recognition motif (Malhotra et al. 1996xMalhotra, A, Severinova, E, and Darst, S.A. Cell. 1996; 87: 127–136Abstract | Full Text | Full Text PDF | PubMed | Scopus (239)See all ReferencesMalhotra et al. 1996), have been determined. Such advances in the understanding of RNAP structure should facilitate elucidation of the more complex activation and repression mechanisms.In the case of σ70, this structural information in conjunction with the finding that σ plays an important role in directing and stabilizing promoter melting is likely to shed light on the mechanism of action of at least some activators that mediate their effects through σ. Since −10 region recognition involves base-specific contacts between the σ subunit and the melted nontemplate strand (Roberts and Roberts 1996xRoberts, C.W and Roberts, J.W. Cell. 1996; 86: 495–501Abstract | Full Text | Full Text PDF | PubMed | Scopus (107)See all ReferencesRoberts and Roberts 1996), it is possible that regulators that interact with σ may, in some cases, stabilize a conformation that favors the formation of these contacts, rather than merely stabilizing the binding of the −35 region recognition domain. Unfortunately, there is as of yet no high resolution structural information about the portion of σ that binds the promoter −35 region, the apparent target of a number of activators that bind in the immediate vicinity of the −35 box. Whether or not the effects of such activator–σ interactions can be transmitted through the structure of σ to the −10 region recognition motif remains to be learned.As the structural analysis of RNAP proceeds, it will be particularly informative to study complexes containing a DNA-bound regulator together with a relevant portion of RNAP. Finally, structural information about the catalytic subunits of RNAP are likely to enhance our understanding of how some activators that contact these subunits work.

CarD is an essential regulator of rRNA transcription required for Mycobacterium tuberculosis persistence.

Mycobacterium tuberculosis is arguably the worlds most successful infectious agent because of its ability to control its own cell growth within the host. Bacterial growth rate is closely coupled to rRNA transcription, which in E. coli is regulated through DksA and (p)ppGpp. The mechanisms of rRNA transcriptional control in mycobacteria, which lack DksA, are undefined. Here we identify CarD as an essential mycobacterial protein that controls rRNA transcription. Loss of CarD is lethal for mycobacteria in culture and during infection of mice. CarD depletion leads to sensitivity to killing by oxidative stress, starvation, and DNA damage, accompanied by failure to reduce rRNA transcription. CarD can functionally replace DksA for stringent control of rRNA transcription, even though CarD associates with a different site on RNA polymerase. These findings highlight a distinct molecular mechanism for regulating rRNA transcription in mycobacteria that is critical for M. tuberculosis pathogenesis.

Crystal Structure of the λ Repressor C-Terminal Domain Provides a Model for Cooperative Operator Binding

Abstract Interactions between transcription factors bound to separate operator sites commonly play an important role in gene regulation by mediating cooperative binding to the DNA. However, few detailed structural models for understanding the molecular basis of such cooperativity are available. The c I repressor of bacteriophage λ is a classic example of a protein that binds to its operator sites cooperatively. The C-terminal domain of the repressor mediates dimerization as well as a dimer–dimer interaction that results in the cooperative binding of two repressor dimers to adjacent operator sites. Here, we present the x-ray crystal structure of the λ repressor C-terminal domain determined by multiwavelength anomalous diffraction. Remarkably, the interactions that mediate cooperativity are captured in the crystal, where two dimers associate about a 2-fold axis of symmetry. Based on the structure and previous genetic and biochemical data, we present a model for the cooperative binding of two λ repressor dimers at adjacent operator sites.

Region 4 of σ as a target for transcription regulation

Bacterial σ factors play a key role in promoter recognition, making direct contact with conserved promoter elements. Most σ factors belong to the σ70 family, named for the primary σ factor in Escherichia coli. Members of the σ70 family typically share four conserved regions and, here, we focus on region 4, which is directly involved in promoter recognition and serves as a target for a variety of regulators of transcription initiation. We review recent advances in the understanding of the mechanism of action of regulators that target region 4 of σ.

A Bacterial Two-Hybrid System Based on Transcription Activation

We describe the use of a bacterial two-hybrid system for the study of protein-protein interactions in Escherichia coli. This system is based on transcription activation and involves the synthesis of two fusion proteins within the bacterial cell whose interaction stimulates transcription of a reporter gene. Specifically, one of the fusion proteins can function as a transcription activator when its interaction partner is fused to a subunit of the bacterial RNA polymerase. This bacterial two-hybrid system has been used to study a number of interacting proteins from both prokaryotes and eukaryotes, and can be used to find interacting proteins from complex protein libraries.

Structure of a Ternary Transcription Activation Complex

The cI protein of bacteriophage lambda (lambdacI) activates transcription by binding a DNA operator just upstream of the promoter and interacting with the RNA polymerase sigma subunit domain 4 (sigma(4)). We determined the crystal structure of the lambdacI/sigma(4)/DNA ternary complex at 2.3 A resolution. There are no conformational changes in either protein, which interact through an extremely small interface involving at most 6 amino acid residues. The interactions of the two proteins stabilize the binding of each protein to the DNA. The results provide insight into how activators can operate through a simple cooperative binding mechanism but affect different steps of the transcription initiation process.