rRNA Promoters as Targets for Transcription Factors: Structural and Functional Studies of PhERI and CarD

Transcription, the process of copying information encoded in DNA into RNA, to facilitate the expression of encoded proteins, is a central process in all living organisms. The expression and repression of subsets of genes allows different cell types in an organism to maintain diverse physiological roles and permits individual cells to respond to various environmental stimuli. Transcription in prokaryotic cells is performed by a single macromolecular complex, RNA polymerase. In rapidly growing cells with abundant resources, prokaryotic RNA polymerase is mostly located at ribosomal RNA (rRNA) promoters, actively transcribing the large, structured RNAs required for protein translation. As resources become more scarce, RNA polymerase directly responds to cellular signals that lead to the repression of rRNA transcription. This regulation has long been thought to be driven primarily by small molecule effectors that signal scarcity. In this thesis, I will report work done on two transcription factors in prokaryotes that regulate RNA polymerase activity at rRNA promoters. The Staphylococcus aureus (Sau) Phage G1 protein PhERI (previously ORF67), was previously described as a general RNA polymerase inhibitor. PhERI expression in Sau cells inhibits cell growth, which could have therapeutic potential against this deadly pathogen. I describe the structure of PhERI bound to Sau σ A 4, the region of RNA polymerase to which it binds. While PhERI interacts with RNAP through σ, I show that RNA polymerase activity at most -10/-35 promoters is not affected. Structural, biochemical and genomic approaches demonstrate that PhERI interacts with σA 4 near the -35 element of all promoters, but blocks the binding of an additional RNA polymerase subunit, the α-CTD, to UP-element DNA sequences. PhERI therefore only inhibits RNA polymerase activity at promoters requiring UP-element activation, most notably the rRNA promoters. This work not only delineates the mechanism of PhERI but also describes novel -10/-35 promoters in Staphlococcus aureus, defines rRNA promoters in this organism for the first time, and shows UP-element activation is required for rRNA transcription. The mycobacterial protein CarD is known to interact with RNA polymerase, but its impact on transcription directly at promoters has not been described. The structure of the Thermus Thermophilus CarD was solved in the lab, allowing a model for the interaction between CarD and RNA polymerase to be built. I show that CarD stimulates RNA polymerase activity at rRNA promoters, but not all promoters, by directly stabilizing the RNA polymerase open complex on promoter DNA. These two proteins both exploit unique parameters of RNA polymerase at rRNA promoters to specifically regulate RNA polymerase activity at these functionally important promoters

Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes

Saccharomyces cerevisiae encodes two distinct Pif1-family helicases – Pif1 and Rrm3 – which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterize the roles of Pif1 helicases in replisome progression and lagging-strand synthesis in S. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulate strand-displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes. Consistent with this observation, we find that head-on collisions between tRNA transcription and replication are under-represented in the S. cerevisiae genome. We demonstrate that tRNA-mediated arrest is R-loop independent, and propose that replisome arrest and DNA damage are mechanistically separable

A new efficient trial design for assessing reliability of ankle-brachial index measures by three different observer groups

BACKGROUND: The usual method of assessing the variability of a measure such as the ankle brachial index (ABI) as a function of different observer groups is to obtain repeated measurements. Because the number of possible observer-subject combinations is impractically large, only a few small studies on inter- and intraobserver variability of ABI measures have been carried out to date. The present study proposes a new and efficient study design. This paper describes the study methodology. METHODS: Using a partially balanced incomplete block design, six angiologists, six primary-care physicians and six trained medical office assistants performed two ABI measurements each on six individuals from a group of 36 unselected subjects aged 65–70 years. Each test subject is measured by one observer from each of the three observer groups, and each observer measures exactly six of the 36 subjects in the group. Each possible combination of two observers occurs exactly once per patient and is not repeated on a second subject. The study involved four groups of 36 subjects (144), plus standbys. RESULTS: The 192 volunteers present at the study day were similar in terms of demographic characteristics and vascular risk factors: mean age 68.6 ± 1.7; mean BMI 29.1 ± 4.6; mean waist-hip ratio 0.92 ± 0.09; active smokers 12%; hypertension 60.9%; hypercholesterolemia 53.4%; diabetic 17.2%. A complete set of ABI measurements (three observers performing two Doppler measurements each) was obtained from 108 subjects. From all other subjects at least one ABI measurement was obtained. The mean ABI was 1.08 (± 0.13), 15 (7.9%) volunteers had an ABI <0.9, and none had an ABI >1.4, i.e. a ratio that may be associated with increased stiffening of the arterial walls. CONCLUSION: This is the first large-scale study investigating the components of variability and thus reliability in ABI measurements. The advantage of the new study design introduced here is that only one sixth of the number of theoretically possible measurements is required to obtain information about measurement errors. Bland-Altman plots show that there are only small differences and no systematic bias between the observers from three occupational groups with different training backgrounds

Intraspecific Body Size Frequency Distributions of Insects

Although interspecific body size frequency distributions are well documented for many taxa, including the insects, intraspecific body size frequency distributions (IaBSFDs) are more poorly known, and their variation among mass-based and linear estimates of size has not been widely explored. Here we provide IaBSFDs for 16 species of insects based on both mass and linear estimates and large sample sizes (n≥100). In addition, we review the published IaBSFDs for insects, though doing so is complicated by their under-emphasis in the literature. The form of IaBSFDs can differ substantially between mass-based and linear measures. Nonetheless, in non-social insects they tend to be normally distributed (18 of 27 species) or in fewer instances positively skewed. Negatively skewed distributions are infrequently reported and log transformation readily removes the positive skew. Sexual size dimorphism does not generally cause bimodality in IaBSFDs. The available information on IaBSFDs in the social insects suggests that these distributions are usually positively skewed or bimodal (24 of 30 species). However, only c. 15% of ant genera are polymorphic, suggesting that normal distributions are probably more common, but less frequently investigated. Although only 57 species, representing seven of the 29 orders of insects, have been considered here, it appears that whilst IaBSFDs are usually normal, other distribution shapes can be found in several species, though most notably among the social insects. By contrast, the interspecific body size frequency distribution is typically right-skewed in insects and in most other taxa

A Systems Engineering Methodology for Information Systems

Complex information systems are often developed without systematic consideration of architectural alternatives partially because systems engineers have lacked a methodology for performing quantitative trade studies of networked systems of sensors, processors, and communications systems. In this paper an approach is discussed for analyzing time-critical information systems and performing systems trades. Information systems are described in terms of design factors with discrete factor levels. Object-oriented models are constructed of the information systems and simulations are run to obtain system measures of performance. Design of experiments is used to drastically reduce the number of models required. The approach is illustrated for an example combat identification information system

A theory of software project management and PROMOL: a project management modeling language

Effective software project management is the key to successful completion of IT software projects. A positive theory of software project management helps to illuminate the path to effective management. Here, we introduce a simple, yet powerful, software project management theory that helps us to understand the conditions and drivers that lead to functional and dysfunctional project behavior. We identify a set of criteria for assessing current and future modeling tools. Finally, we introduce a formal and visual modeling language for management of software projects.Approved for public release; distribution is unlimited

A Systems Engineering Methodology for Analyzing Systems of Systems Using the Systems Modeling Language (SysML)

A systems engineering methodology for analyzing a system of systems (SoS) elucidated in this paper involves the use of process modeling, modeling of the SoS with the systems modeling language (SysML), and subsequent conversion of the resulting SySML model into an end-to-end system of systems executable object-oriented simulation model. A process is a series of actions undertaken by a system or a system of systems to produce one or more end results, typically products and services. The SysML extends and customizes UML 2 to support systems engineering activities in engineering of complex systems. The methodology represents part of our on-going research at the Naval Postgraduate School (NPS) in establishing an integrated methodology for SoS architecting and engineering to be used in industry. In this paper we will explain the methodology and emphasize the correspondence between the SysML representation of a conceptual SoS and its executable model (via Extend TM); we will also illustrate the methodology with an exploratory application to analysis of a coalition U.S.-Singapore SoS architecture employed to counter terrorism emanating from the maritime domain. Our future work is aimed at extending the methodology and improving its rigor and trying it to ontological engineering.This work was supported in party by a contract with the OUSD (AT&L) through System of Systems Engineering Center of Excellence (SoSECE)

Connections in System of Systems

Systems of systems are becoming more important in today’s global endeavors. In this paper use of model-based systems engineering is examined from the viewpoint of understanding connections, that is the transfer of items between system of systems. The conclusion is that systems engineering models must include executable models in order to best understand the effects of transfer of items in system of systems