Global kinetic mechanism describing single nucleotide incorporation for RNA polymerase I reveals fast UMP incorporation.

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I. To this end, we report a transient state kinetic interrogation of AMP, CMP, GMP, and UMP incorporations catalyzed by Pol I. We found that Pol I uses one kinetic mechanism to incorporate all nucleotides. However, we found that UMP incorporation is faster than AMP, CMP, and GMP additions. Further, we found that endonucleolytic removal of a dimer from the 3' end was fastest when the 3' terminal base is a UMP. It has been previously shown that both downstream and upstream template sequence identity impacts the kinetics of nucleotide addition. The results reported here show that the incoming base identity also impacts the magnitude of the observed rate limiting step.

Reversible Kinetics in Multi-nucleotide Addition Catalyzed by S. cerevisiae RNA polymerase II Reveal Slow Pyrophosphate Release.

Eukaryotes express at least three nuclear DNA dependent RNA polymerases (Pols). Pols I, II, and III synthesize ribosomal (r) RNA, messenger (m) RNA, and transfer (t) RNA, respectively. Pol I and Pol III have intrinsic nuclease activity conferred by the A12.2 and C11 subunits, respectively. In contrast, Pol II requires the transcription factor (TF) IIS to confer robust nuclease activity. We recently reported that in the absence of the A12.2 subunit Pol I reverses bond formation by pyrophosphorolysis in the absence of added PPi, indicating slow PPi release. Thus, we hypothesized that Pol II, naturally lacking TFIIS, would reverse bond formation through pyrophosphorolysis. Here we report the results of transient-state kinetic experiments to examine the addition of nine nucleotides to a growing RNA chain catalyzed by Pol II. Our results indicate that Pol II reverses bond formation by pyrophosphorolysis in the absence of added PPi. We propose that, in the absence of endonuclease activity, this bond reversal may represent kinetic proofreading. Thus, given the hypothesis that Pol I evolved from Pol II through the incorporation of general transcription factors, pyrophosphorolysis may represent a more ancient form of proofreading that has been evolutionarily replaced with nuclease activity.

Dietary Uptake of Highly Hydrophobic Chemicals by Rainbow Trout (Oncorhynchus Mykiss).

Rainbow trout (Oncorhynchus mykiss) was exposed through the diet to a mixture of non-ionic organic chemicals for 28 d, followed by a depuration phase, in accordance with OECD method 305. The mixture included hexachlorobenzene (HCB), 2,2',5,5'-tetrachlorobiphenyl (PCB-52), 2,2',5,5'-hexachlorobiphenyl (PCB-153), decachlorobiphenyl (PCB-209), decabromodiphenyl ether (BDE209), decabromodiphenyl ethane (DBDPE), bis-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBPH), perchloro-p-terphenyl (p-TCP), perchloro-m-terphenyl (m-TCP), and perchloro-p-quaterphenyl (p-QTCP), the latter six of which are considered highly hydrophobic based on n-octanol/water partition coefficients (KOW) greater than 108. All chemicals had first-order uptake and elimination kinetics except p-QTCP, whose kinetics could not be verified due to limitations of analytical detection in the elimination phase. For HCB and PCBs, the growth-corrected elimination rates (k2g), assimilation efficiencies (α), and biomagnification factors (BMFL) corrected for lipid content compared well with literature values. For the highly hydrophobic chemicals, elimination rates were faster than the rates for HCB and PCBs, and α's and BMFLs were much lower than those of HCB and PCBs, i.e., ranging from 0.019 to 2.8%, and from 0.000051 to 0.023 (g-lipid/g-lipid), respectively. As a result, the highly hydrophobic organic chemicals were found be much less bioavailable and bioaccumulative than HCB and PCBs. Based on the current laboratory dietary exposures, none of the highly hydrophobic substances would be expected to biomagnify, but Trophic Magnification Factors (TMFs) > 1 have been reported from field studies for TBPH and DBDPE. Additional research is needed to understand and reconcile the apparent inconsistencies in these two lines of evidence for bioaccumulation assessment.

The A12.2 Subunit Plays an Integral Role in Pyrophosphate Release of RNA Polymerase I.

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA), which is the first and rate-limiting step in ribosome biosynthesis. A12.2 (A12) is a critical subunit of Pol I that is responsible for activating Pol I's exonuclease activity. We previously reported a kinetic mechanism for single-nucleotide incorporation catalyzed by Pol I lacking the A12 subunit (ΔA12 Pol I) purified from S. cerevisae and revealed that ΔA12 Pol I exhibited much slower incorporation compared to Pol I. However, it is unknown if A12 influences each nucleotide incorporation in the context of transcription elongation. Here, we show that A12 contributes to every repeating cycle of nucleotide addition and that deletion of A12 results in an entirely different kinetic mechanism compared to WT Pol I. We found that instead of one irreversible step between each nucleotide addition cycle, as reported for wild type (WT) Pol I, the ΔA12 variant requires one reversible step to describe each nucleotide addition. Reversibility fundamentally requires slow PPi release. Consistently, we show that Pol I is more pyrophosphate (PPi) concentration dependent than ΔA12 Pol I. This observation supports the model that PPi is retained in the active site of ΔA12 Pol I longer than WT Pol I. These results suggest that A12 promotes PPi release, revealing a larger role for the A12.2 subunit in the nucleotide addition cycle beyond merely activating exonuclease activity.

RNA Polymerase I Is Uniquely Vulnerable to the Small-Molecule Inhibitor BMH-21.

Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols' elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I.

Estimating n-octanol-water partition coefficients for neutral highly hydrophobic chemicals using measured n-butanol-water partition coefficients.

Direct measurement of the n-octanol partition coefficients (KOW) for highly hydrophobic organic chemicals is extremely difficult because of the extremely low concentrations present in the water phase. n-Butanol/water partition coefficients (KBW) are generally much lower than KOW due to the increased solubility of solute in the alcohol saturated aqueous phase, and therefore become easier to measure. We measured the KBW for 25 neutral organic chemicals having measured log KOWs ranging from 2 to 9 and 4 additional highly hydrophobic chemicals, with unmeasured KOWs, having estimated log KOWs ranging from 6 to 18. The measured log KBW and log KOW values were linearly related, r2 = 0.978, and using the regression developed from the data, KOWs were predicted for the 4 highly hydrophobic chemicals with unmeasured KOWs. The resulting predictions were orders of magnitude lower than those predicted by a variety of computational models and suggests the estimates of KOW in the literature for highly hydrophobic chemicals (i.e., log KOW greater than 10) are likely incorrect by several orders of magnitude.

Canadian home parenteral nutrition (HPN) registry: validation and patient outcomes.

BACKGROUND: In Canada, there are an estimated 400 home parenteral nutrition (HPN) patients. In 2006, a registry was created to gather patient outcome information. The aim of this study was to validate the registry and report on HPN patient outcomes. METHODS: Several demographic, clinical parameters were collected. For the validation, paired t test and intraclass correlation coefficient (ICC) were used to assess agreement between repeat entries. For the outcome report, paired t test was used to assess changes, and survival analysis was performed using the Kaplan-Meier method. Results are expressed as mean ± SEM. RESULTS: On validation, there was high correlation/agreement (P < .05) for most parameters except vascular access/line sepsis, liver disease (ultrasound, biopsy, diagnoses), and hospitalizations. For the outcome report, 96 patients had their data entered at 2.24 ± 0.11 years after baseline. Over the period, there was a significant reduction in PN calories (P = .001) and proteins (P < .001). There were no significant changes in nutrition parameters and laboratory results except for lower platelet counts (P = .028), lower plasma potassium (P = .030), and a trend toward an increase in bilirubin from 19.29 ± 4.65 to 29.06 ± 8.73 µmol/L (P = .071). The QOL decreased significantly over time (P < .001) and the survival on HPN was 17.67 ± 1.89 years. CONCLUSIONS: The registry is a valid tool to assess several clinical parameters. On follow-up, HPN patients maintain good nutrition status while PN is reduced but do have a reduced quality of life.

The effect of day care exposure on the risk of developing type 1 diabetes: a meta-analysis of case-control studies.

OBJECTIVE: Exposure to infections in infancy or childhood may be important in the pathogenesis of type 1 diabetes, but a protective role has also been suggested. We tested the hypothesis that increased early contact with infectious agents, measured by day care exposure, would decrease the risk of type 1 diabetes in childhood. RESEARCH DESIGN AND METHODS: We conducted a systematic review of case-control studies. Meta-analysis was performed to combine results, assess for heterogeneity, and explore variation in study design. RESULTS: Several generally well-designed case-control studies show a statistically significant protective effect of day care on type 1 diabetes. However, meta-analysis revealed too much heterogeneity to accept the overall synthesis results and none of the studies used prerecorded data. Day care does seem to have a protective effect in the subgroup of children who will be diagnosed with type 1 diabetes before the age of 5 years (odds ratio = 0.6, 95% CI 0.5-0.8); however, this result is based on only two studies. CONCLUSIONS: Recall bias is one alternate explanation for these data; confirmation using prerecorded data is required. Such data could be prospectively measured in cohort studies of children at risk. We also suggest that information about day care attendance be measured in randomized trials of agents for the prevention of type 1 diabetes, as day care exposure could potentially modify the effect of the preventive agent.