To elucidate the cellular role of the heterotrimeric G protein G o , we have taken a molecular genetic approach in Caenorhabditis elegans. We screened for suppressors of activated GOA-1 (G o ␣) that do not simply decrease its expression and found mutations in only two genes, sag-1 and eat-16. Animals defective in either gene display a hyperactive phenotype similar to that of goa-1 loss-of-function mutants. Double-mutant analysis indicates that both sag-1 and eat-16 act downstream of, or parallel to, G o ␣ and negatively regulate EGL-30 (G q ␣) signaling. eat-16 encodes a regulator of G protein signaling (RGS) most similar to the mammalian RGS7 and RGS9 proteins and can inhibit endogenous mammalian G q /G 11 in COS-7 cells. Animals defective in both sag-1 and eat-16 are inviable, but reducing function in egl-30 restores viability, indicating that the lethality of the eat-16; sag-1 double mutant is due to excessive G q ␣ activity. Analysis of these mutations indicates that the G o and G q pathways function antagonistically in C. elegans, and that G o ␣ negatively regulates the G q pathway, possibly via EAT-16 or SAG-1. We propose that a major cellular role of G o is to antagonize signaling by G q .
Eukaryotes have three distinct RNA polymerases that catalyze transcription of nuclear genes. RNA polymerase II is responsible for transcribing nuclear genes encoding the messenger RNAs and several small nuclear RNAs. Like RNA polymerases I and III, polymerase II cannot recognize its target promoter directly and initiate transcription without accessory factors. Instead, this large multisubunit enzyme relies on general transcription factors and transcriptional activators and coactivators to regulate transcription from class II promoters. X-ray crystallography and nuclear magnetic resonance spectroscopy have been used to study complexes of general transcription factors and transcriptional activators with their specific DNA targets. This work has provided important structural insights into transcription initiation by polymerase II and the more general problem of DNA sequence recognition.
The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) sigma(70) factor. Rsd plays a role in alternative sigma factor-dependent transcription by biasing the competition between sigma(70) and alternative sigma factors for the available core RNAP. Here, we determined the 2.6 A-resolution X-ray crystal structure of Rsd bound to sigma(70) domain 4 (sigma(70)(4)), the primary determinant for Rsd binding within sigma(70). The structure reveals that Rsd binding interferes with the two primary functions of sigma(70)(4), core RNAP binding and promoter -35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the sigma(70)(4)-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between sigma(70)(4) binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.
Gbeta(5) functions in vivo complexed with GGL-containing RGS proteins. In the absence of Gbeta(5), these RGS proteins have little or no function. The formation of RGS-Gbeta(5) complexes is required for the expression or stability of both the RGS and Gbeta(5) proteins. Appropriate RGS-Gbeta(5) complexes regulate both Galpha(o) and Galpha(q) proteins in vivo.
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