Members of the SR family of proteins, can collaborate with U1 snRNP in the recognition of 5' splice sites in pre-messenger RNAs. We have previously shown that purified U1 snRNP and ASF/SF2 form a ternary complex with pre-mRNA, which is dependent on a functional 5' splice site. In this manuscript we dissect the requirements for the formation of this complex. Sequences in the pre-mRNA, domains in ASF/SF2 and components of the U1 snRNP particle are shown to be required for complex formation. We had shown that sequences at the 5' splice site of PIP7. A are necessary and now we show these are sufficient for complex formation. Furthermore, we show that one functional RNA binding domain and the RS domain are both required for ASF/SF2 to participate in complex formation. The RNA binding domains were redundant in this assay, suggesting that either domain can interact with the pre-messenger RNA. Finally, our experiments show no function for the U1-specific A protein in complex formation, whereas a function for U1-specific C protein was strongly suggested. The study of the earliest interactions between pre-mRNA and splicing factors suggests a model for 5' splice site recognition.

To terminate transcription in E. coli, Rho protein binds an RNA loading site on the nascent transcript, translocates 5'--> 3' along the RNA in an ATP-driven process, and, upon reaching the transcription elongation complex, brings about RNA release. Thus, the Rho-dependent termination process can be viewed, in part, as a kinetic competition between the rate of transcript elongation by RNA polymerase (RNAP) and the rate of Rho translocation along the nascent transcript. In the context of this model, NusG, which is an essential E. coli protein, regulates Rho-dependent termination in an apparently paradoxical way, increasing the rate of transcription elongation of E. coli RNAP in the absence of Rho while also shifting the sites of Rho-dependent termination upstream on the template. Here we investigate the regulation of Rho-dependent termination by NusG. Analytical ultracentrifugation was used to establish the existence of a stable complex of NusG and Rho and to demonstrate a stoichiometry of one NusG monomer per Rho hexamer. Surface plasmon resonance was used to examine the kinetics of the formation and dissociation of the NusG-Rho complex, yielding an association rate constant (k(on)) of 2.8 (+/-0.8) x 10(5) M(-)(1) s(-)(1), a dissociation rate constant (k(off)) of 3.9 (+/-0.7) x 10(-)(3) s(-)(1), and a calculated equilibrium (dissociation) constant (K(d)) of 1.5 (+/-0.3) x 10(-)(8) M. The apparent stability of the NusG-Rho complex is insensitive to changes in salt (potassium acetate) concentration between 0.05 and 0.15 M. The translocation and transcription termination activities of Rho at saturating NusG concentrations were, however, both sensitive to salt concentration over this range, suggesting that these activities do not directly reflect the stability of the NusG-Rho complex. Rho-dependent termination could be demonstrated for transcription complexes in which E. coli RNAP had been substituted by either bacteriophage SP6 or T7 RNAP. NusG, however, was not active in transcription termination assays with either of these phage RNAPs. Thus, we conclude that NusG modulates Rho-dependent termination by interacting specifically with the RNAP of the E. coli elongation complex to render the complex more susceptible to the termination activity of Rho.

The decision to elongate or terminate the RNA chain at specific DNA template positions during transcription is kinetically regulated, but the methods used to measure the rates of these processes have not been sufficiently quantitative to permit detailed mechanistic analysis of the steps involved. Here, we use surface plasmon resonance (SPR) technology to monitor RNA transcription by Escherichia coli RNA polymerase (RNAP) in solution and in real time. We show that binding of RNAP to immobilized DNA templates to form active initiation or elongation complexes can be resolved and monitored by this method, and that changes during transcription that involve the gain or loss of bound mass, including the release of the sigma factor during the initiation-elongation transition, the synthesis of the RNA transcript, and the release of core RNAP and nascent RNA at intrinsic terminators, can all be observed. The SPR method also permits the discrimination of released termination products from paused and other intermediate complexes at terminators. We have used this approach to show that the rate constant for transcript release at intrinsic terminators tR2 and tR is Ϸ2-3 s ؊1 and that the extent of release at these terminators is consistent with known termination efficiencies. Simulation techniques have been used to fit the measured parameters to a simple kinetic model of transcription and the implications of these results for transcriptional regulation are discussed.intrinsic termination ͉ kinetic modeling ͉ RNA polymerase binding ͉ elongation complex dissociation ͉ sigma release R NA transcription is a central event in gene expression and is tightly controlled during all of the phases of synthesis of the full-length transcript, including initiation, elongation, and termination. Misregulation during any of these processes can result in aberrant gene expression, compromise survival of singlecelled creatures, and lead to disease in higher organisms. Although termination is required to stop transcript elongation at the ends of genes, this process also plays important regulatory roles at intermediate template positions during transcription elongation (1-4). The transcription of many essential genes across all phyla, including humans, is controlled in the postinitiation phase by termination events that are mediated by signals in the nascent RNA or DNA and are often further modulated by protein cofactors (5, 6). The fundamental structure of the nucleic acid scaffold that constitutes the core of the transcription elongation complex (TEC) is conserved in all organisms. Hence, the well described Escherichia coli TEC serves as an excellent model system to study basic aspects of transcription termination and its regulation.Pathways that can potentially compete with elongation at each template position during transcription include pyrophosphorolysis (the chemical reverse of the NTP addition process), entrance into arrested or editing states, and termination (4, 7-10). Sequence-specific pausing of the TEC can also occur, often as a prelude...

No abstract

Aminoacyl-tRNA synthetases (ARSs) enhance the fidelity of protein synthesis through multiple mechanisms, including hydrolysis of the adenylate and cleavage of misacylated tRNA. Alanyl-tRNA synthetase (AlaRS) limits misacylation with glycine and serine by use of a dedicated editing domain, and a mutation in this activity has been genetically linked to a mouse model of a progressive neurodegenerative disease. Using the free standing P. horikoshii AlaX editing domain complexed with serine as a model and both Ser-tRNAAla and Ala-tRNAAla as substrates, the deacylation activities of the wild type and five different E. coli AlaRS editing site substitution mutants were characterized. The wild type AlaRS editing domain deacylated Ser-tRNAAla with a kcat/KM of 6.6 × 105 M−1 s−1, equivalent to a rate enhancement of 6000 over the rate of enzyme-independent deacylation, but only 12.2-fold greater than the rate with Ala-tRNAAla. While the E664A and T567G substitutions only minimally decreased kcat/KM, Q584H, I667E, and C666A AlaRS were more compromised in activity, with decreases in kcat/KM in the range of 6-, 7.3-, and 15-fold. C666A AlaRS was 1.4-fold more active on Ala-tRNAAla relative to Ser-tRNAAla, providing the only example of a true reversal of substrate specificity and highlighting a potential role of the coordinated zinc in editing substrate specificity. Along with the potentially serious physiological consequences of serine mis-incorporation, the relatively modest specificity of the AlaRS editing domain may provide a rationale for the widespread phylogenetic distribution of AlaX free standing editing domains, thereby contributing a further mechanism to lower concentrations of misacylated tRNAAla.