RESUMEN
BACKGROUND: Previously, we showed that adaptive substitutions in one of the three promoters of the bacteriophage φX174 improved fitness at high-temperature by decreasing transcript levels three- to four-fold. To understand how such an extreme change in gene expression might lead to an almost two-fold increase in fitness at the adaptive temperature, we focused on stages in the life cycle of the phage that occur before and after the initiation of transcription. For both the ancestral strain and two single-substitution strains with down-regulated transcription, we measured seven phenotypic components of fitness (attachment, ejection, eclipse, virion assembly, latent period, lysis rate and burst size) during a single cycle of infection at each of two temperatures. The lower temperature, 37°C, is the optimal temperature at which phages are cultivated in the lab; the higher temperature, 42°C, exerts strong selection and is the condition under which these substitutions arose in evolution experiments. We augmented this study by developing an individual-based stochastic model of this same life cycle to explore potential explanations for our empirical results. RESULTS: Of the seven fitness parameters, three showed significant differences between strains that carried an adaptive substitution and the ancestor, indicating the presence of pleiotropy in regulatory evolution. 1) Eclipse was longer in the adaptive strains at both the optimal and high-temperature environments. 2) Lysis rate was greater in the adaptive strains at the high temperature. 3) Burst size for the mutants was double that of the ancestor at the high temperature, but half that at the lower temperature. Simulation results suggest that eclipse length and latent period variance can explain differences in burst sizes and fitness between the mutant and ancestral strains. CONCLUSIONS: Down-regulating transcription affects several steps in the phage life cycle, and all of these occur after the initiation of transcription. We attribute the apparent tradeoff between delayed progeny production and faster progeny release to improved host resource utilization at high temperature.
Asunto(s)
Bacteriófago phi X 174/fisiología , Bacteriófago phi X 174/genética , Bacteriófago phi X 174/crecimiento & desarrollo , Escherichia coli/virología , Regulación Viral de la Expresión Génica , Aptitud Genética , Modelos Biológicos , Mutación , Transcripción Genética , Ensamble de VirusRESUMEN
To investigate how high temperature affects viral transcription, the absolute amounts of mRNA for six bacteriophage φX174 genes were compared at 37 °C and 42 °C using Q-PCR. At 37 °C, mRNA levels for all genes were consistent with previous studies, but at 42 °C mRNA levels for four genes were significantly different from levels at 37 °C. Transcript levels were higher for genes B and D; the promoter before gene B appears to be up-regulated at high temperature. Levels for genes F and G were reduced at high temperature, possibly due to increased efficiency of the transcription termination signal immediately upstream of gene F. These functional changes in φX174 gene regulation at high temperature have not been described previously. Studies of phage evolution at high temperatures indicate that this difference in transcript levels is subject to adaptation.
Asunto(s)
Bacteriófagos/metabolismo , Proteínas Virales/genética , Secuencia de Bases , Unión Proteica , ARN Mensajero/metabolismo , Factor sigma/genética , Factor sigma/metabolismo , Temperatura , Transcripción Genética , Proteínas Virales/metabolismoRESUMEN
Developing a comprehensive description of the equilibrium structural ensembles for intrinsically disordered proteins (IDPs) is essential to understanding their function. The p53 transactivation domain (p53TAD) is an IDP that interacts with multiple protein partners and contains numerous phosphorylation sites. Multiple techniques were used to investigate the equilibrium structural ensemble of p53TAD in its native and chemically unfolded states. The results from these experiments show that the native state of p53TAD has dimensions similar to a classical random coil while the chemically unfolded state is more extended. To investigate the molecular properties responsible for this behavior, a novel algorithm that generates diverse and unbiased structural ensembles of IDPs was developed. This algorithm was used to generate a large pool of plausible p53TAD structures that were reweighted to identify a subset of structures with the best fit to small angle X-ray scattering data. High weight structures in the native state ensemble show features that are localized to protein binding sites and regions with high proline content. The features localized to the protein binding sites are mostly eliminated in the chemically unfolded ensemble; while, the regions with high proline content remain relatively unaffected. Data from NMR experiments support these results, showing that residues from the protein binding sites experience larger environmental changes upon unfolding by urea than regions with high proline content. This behavior is consistent with the urea-induced exposure of nonpolar and aromatic side-chains in the protein binding sites that are partially excluded from solvent in the native state ensemble.
Asunto(s)
Pliegue de Proteína , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Cromatografía en Gel , Humanos , Hidrodinámica , Luz , Modelos Moleculares , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Urea/metabolismo , Difracción de Rayos XRESUMEN
BACKGROUND: Gene regulation plays a central role in the adaptation of organisms to their environments. There are many molecular components to gene regulation, and it is often difficult to determine both the genetic basis of adaptation and the evolutionary forces that influence regulation. In multiple evolution experiments with the bacteriophage ÏX174, adaptive substitutions in cis-acting regulatory sequences sweep through the phage population as the result of strong positive selection at high temperatures that are non-permissive for laboratory-adapted phage. For one cis-regulatory region, we investigate the individual effects of four adaptive substitutions on transcript levels and fitness for phage growing on three hosts at two temperatures. RESULTS: The effect of the four individual substitutions on transcript levels is to down-regulate gene expression, regardless of temperature or host. To ascertain the conditions under which these substitutions are adaptive, fitness was measured by a variety of methods for several bacterial hosts growing at two temperatures, the control temperature of 37°C and the selective temperature of 42°C. Time to lysis and doublings per hour indicate that the four substitutions individually improve fitness over the ancestral strain at high temperature independent of the bacterial host in which the fitness was measured. Competition assays between the ancestral strain and either of two mutant strains indicate that both mutants out-compete the ancestor at high temperature, but the relative frequencies of each phage remain the same at the control temperature. CONCLUSIONS: Our results strongly suggest that gene transcription plays an important role in influencing fitness in the bacteriophage ÏX174, and different point mutations in a single cis-regulatory region provided the genetic basis for this role in adaptation to high temperature. We speculate that the adaptive nature of these substitutions is due to the physiology of the host at high temperature or the need to maintain particular ratios of phage proteins during capsid assembly. Our investigation of regulatory evolution contributes to interpreting genome-level assessments of regulatory variation, as well as to understanding the molecular basis of adaptation.
Asunto(s)
Bacteriófago phi X 174/genética , Calor , Selección Genética , Transcripción Genética , Adaptación Biológica/genética , Regulación Viral de la Expresión Génica , Aptitud Genética , Mutagénesis Sitio-Dirigida , Mutación Puntual , ARN Viral/genéticaRESUMEN
Internuclear distances derived from paramagnetic relaxation enhancement (PRE) data were used to restrain molecular dynamics simulations of the intrinsically unstructured transactivation domain of the tumor suppressor protein, p53. About 1000 structures were simulated using ensemble averaging of replicate molecules to compensate for the inherent bias in the PRE-derived distances. Gyration radii measurements on these structures show that the p53 transactivation domain (p53TAD) is statistically predominantly in a partially collapsed state that is unlike the open structure that is found for p53TAD bound to either the E3 ubiquitin ligase, MDM2, or the 70 kDa subunit of replication protein A, RPA70. Contact regions that potentially mediate the collapse were identified and found to consist of mostly hydrophobic residues. The identified contact regions preferentially place the MDM2 and RPA70 binding regions in close proximity. We show that our simulations thoroughly sample the available range of conformations and that a fraction of the molecules are in an open state that would be competent for binding either MDM2 or RPA70. We also show that the Stokes radius estimated from the average gyration radius of the ensemble is in good agreement with the value determined using size exclusion chromatography. Finally, the presence of a persistent loop localized to a PXP motif was identified. Serine residues flanking the PXP motif become phosphorylated in response to DNA damage, and we postulate that this will perturb the equilibrium population to more open conformations.
Asunto(s)
Activación Transcripcional , Proteína p53 Supresora de Tumor/química , Simulación por Computador , Humanos , Modelos Químicos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estructura Terciaria de Proteína , Marcadores de SpinRESUMEN
Paramagnetic relaxation enhancement (PRE) was used to identify a compact dynamic structure for the intrinsically unstructured transactivation domain of the tumor suppressor protein, p53. Our results show that p53 residues essential for binding to the ubiquitin ligase, MDM2, and the 70 kDa subunit of replication protein A, RPA70, are separated by an average distance of 10-15 A. This result suggests that a more extended member of the ensemble must be populated prior to binding either MDM2 or RPA70. We also show that PRE can be used to detect intermolecular distances between p53 and RPA70.