Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment.

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.

Preparation of RNA Polymerase Complexes for Their Analysis by Single-Particle Cryo-Electron Microscopy.

Recent technological progress revealed new prospects of high-resolution structure determination of macromolecular complexes using cryo-electron microscopy (cryo-EM) . In the field of RNA polymerase (Pol) I research, a number of cryo-EM studies contributed to understanding the highly specialized mechanisms underlying the transcription of ribosomal RNA genes . Despite a broad applicability of the cryo-EM method itself, preparation of samples for high-resolution data collection can be challenging. Here, we describe strategies for the purification and stabilization of Pol I complexes, exemplarily considering advantages and disadvantages of the methodology. We further provide an easy-to-implement protocol for the coating of EM-grids with self-made carbon support films. In sum, we present an efficient workflow for cryo-grid preparation and optimization, including early stage cryo-EM screening that can be adapted to a wide range of soluble samples for high-resolution structure determination .

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor-binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain-containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

Conserved strategies of RNA polymerase I hibernation and activation.

RNA polymerase (Pol) I transcribes the ribosomal RNA precursor in all eukaryotes. The mechanisms 'activation by cleft contraction' and 'hibernation by dimerization' are unique to the regulation of this enzyme, but structure-function analysis is limited to baker's yeast. To understand whether regulation by such strategies is specific to this model organism or conserved among species, we solve three cryo-EM structures of Pol I from Schizosaccharomyces pombe in different functional states. Comparative analysis of structural models derived from high-resolution reconstructions shows that activation is accomplished by a conserved contraction of the active center cleft. In contrast to current beliefs, we find that dimerization of the S. pombe polymerase is also possible. This dimerization is achieved independent of the 'connector' domain but relies on two previously undescribed interfaces. Our analyses highlight the divergent nature of Pol I transcription systems from their counterparts and suggest conservation of regulatory mechanisms among organisms.

DNA origami-based single-molecule force spectroscopy elucidates RNA Polymerase III pre-initiation complex stability.

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.

Suit the action to the word, the word to the action: Hypothetical choices and real decisions in Medicare Part D.

In recent years, consumer choice has become an important element of public policy. One reason is that consumers differ in their tastes and needs, which they can express most easily through their own choices. Elements that strengthen consumer choice feature prominently in the design of public insurance markets, for instance in the United States in the recent introduction of prescription drug coverage for older individuals via Medicare Part D. For policy makers who design such a market, an important practical question in the design phase of such a new program is how to deduce enrollment and plan selection preferences prior to its introduction. In this paper, we investigate whether hypothetical choice experiments can serve as a tool in this process. We combine data from hypothetical and real plan choices, elicited around the time of the introduction of Medicare Part D. We first analyze how well the hypothetical choice data predict willingness to pay and market shares at the aggregate level. We then analyze predictions at the individual level, in particular how insurance demand varies with observable characteristics. We also explore whether the extent of adverse selection can be predicted using hypothetical choice data alone.

Plan selection in Medicare Part D: evidence from administrative data.

We study the Medicare Part D prescription drug insurance program as a bellwether for designs of private, non-mandatory health insurance markets, focusing on the ability of consumers to evaluate and optimize their choices of plans. Our analysis of administrative data on medical claims in Medicare Part D suggests that fewer than 25% of individuals enroll in plans that are ex ante as good as the least cost plan specified by the Plan Finder tool made available to seniors by the Medicare administration, and that consumers on average have expected excess spending of about $300 per year, or about 15% of expected total out-of-pocket cost for drugs and Part D insurance. These numbers are hard to reconcile with decision costs alone; it appears that unless a sizeable fraction of consumers place large values on plan features other than cost, they are not optimizing effectively.

Dynamics of self-rated health and selective mortality.

Self-rated health status (SRHS) is one of the most frequently used health measures in empirical health economics. This article analyzes the first seven waves of the Health and Retirement Study (HRS) and finds that (1) all available lags have decreasing but significant predictive power for current SRHS and (2) SRHS and future mortality are strongly related which leads to a specific selection problem known as survivorship bias. A parsimonious joint model with an autocorrelated latent health component in both the SRHS and the mortality equation is suggested. It is better able to capture the empirical facts than commonly used models including random effects and/or state dependence and better able to correct the survivorship bias than commonly used strategies such as inverse probability weighting.

Who failed to enroll in Medicare Part D, and why? Early results.

Early results on the Medicare Part D prescription drug program, from a survey of people age sixty-five and older who were interviewed just before enrollment started and just after it ended, indicate that Medicare has met its target of 90 percent coverage. Enrollment rates in vulnerable subpopulations-poor health, low income, or cognitive impairment-are almost high enough to offset lower rates of other coverage. However, sizable numbers of elderly people remain uncovered, contrary to their self-interest. Seniors give Part D mixed reviews, and majorities are less satisfied with Medicare and with the government as a result of their experience with this program.

Medicare prescription drug coverage: consumer information and preferences.

We investigate prescription drug use, and information and enrollment intentions for the new Medicare Part D drug insurance program, using a sample of Medicare-eligible subjects surveyed before open enrollment began for this program. We find that, despite the complexity of competing plans offered by private insurers under Part D, a majority of the Medicare population had information on this program and a substantial majority planned to enroll. We find that virtually all elderly, even those with no current prescription drug use, can expect to benefit from enrollment in a Part D Standard plan at the low premiums available in the current market. However, there is a significant risk that many eligible seniors, particularly low-income elderly with poor health or cognitive impairment, will make poor enrollment and plan choices.