RESUMO
Transcription is a tightly regulated, complex, and essential cellular process in all living organisms. Transcription is comprised of three steps, transcription initiation, elongation, and termination. The distinct transcription initiation and termination mechanisms of eukaryotic RNA polymerases I, II, and III (Pols I, II, and III) have long been appreciated. Recent methodological advances have empowered high-resolution investigations of the Pols' transcription elongation mechanisms. Here, we review the kinetic similarities and differences in the individual steps of Pol I-, II-, and III-catalyzed transcription elongation, including NTP binding, bond formation, pyrophosphate release, and translocation. This review serves as an important summation of Saccharomyces cerevisiae (yeast) Pol I, II, and III kinetic investigations which reveal that transcription elongation by the Pols is governed by distinct mechanisms. Further, these studies illustrate how basic, biochemical investigations of the Pols can empower the development of chemotherapeutic compounds.
Assuntos
Tratamento Farmacológico , RNA Polimerase III , RNA Polimerase II , RNA Polimerase I , Saccharomyces cerevisiae , Elongação da Transcrição Genética , Biocatálise/efeitos dos fármacos , Cinética , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Saccharomyces cerevisiae/enzimologia , Elongação da Transcrição Genética/efeitos dos fármacosRESUMO
Eukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale. We analyzed Saccharomyces cerevisiae Pol II incorporation of ATP or an ATP analog, Sp-ATP-α-S. Here we have measured the rate constants governing individual steps of the Pol II transcription cycle in the presence of ATP or Sp-ATP-α-S. These results suggest that Pol II catalyzes nucleotide incorporation by binding the next cognate nucleotide and immediately catalyzes bond formation and bond formation is either followed by a conformational change or pyrophosphate release. By comparing our previously published RNA polymerase I (Pol I) and Pol I lacking the A12 subunit (Pol I ΔA12) results that we collected under the same conditions with the identical technique, we show that Pol II and Pol I ΔA12 exhibit similar nucleotide addition mechanisms. This observation indicates that removal of the A12 subunit from Pol I results in a Pol II like enzyme. Taken together, these data further our collective understanding of Pol II's nucleotide incorporation mechanism and the evolutionary divergence of RNA polymerases across the three domains of life.
Assuntos
Nucleotídeos , RNA Polimerase II , Nucleotídeos/metabolismo , RNA Polimerase II/metabolismo , Cinética , RNA Polimerase I/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Cancer cells are dependent upon an abundance of ribosomes to maintain rapid cell growth and proliferation. The rate-limiting step of ribosome biogenesis is ribosomal RNA (rRNA) synthesis by RNA polymerase I (Pol I). Therefore, a goal of the cancer therapeutic field is to develop and characterize Pol I inhibitors. Here, we elucidate the mechanism of Pol I inhibition by a first-in-class small-molecule BMH-21. To characterize the effects of BMH-21 on Pol I transcription, we leveraged high-resolution in vitro transcription assays and in vivo native elongating transcript sequencing (NET-seq). We find that Pol I transcription initiation, promoter escape, and elongation are all inhibited by BMH-21 in vitro. In particular, the transcription elongation phase is highly sensitive to BMH-21 treatment, as it causes a decrease in transcription elongation rate and an increase in paused Pols on the ribosomal DNA (rDNA) template. In vivo NET-seq experiments complement these findings by revealing a reduction in Pol I occupancy on the template and an increase in sequence-specific pausing upstream of G-rich rDNA sequences after BMH-21 treatment. Collectively, these data reveal the mechanism of action of BMH-21, which is a critical step forward in the development of this compound and its derivatives for clinical use.
Assuntos
DNA Ribossômico , Compostos Heterocíclicos de 4 ou mais Anéis , RNA Polimerase I , Transcrição Gênica , DNA Ribossômico/genética , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Polimerase II/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Eukaryotes express at least three nuclear DNA-dependent RNA polymerases (Pols) responsible for synthesizing all RNA required by the cell. Despite sharing structural homology, they have functionally diverged to suit their distinct cellular roles. Although the Pols have been studied extensively, direct comparison of their enzymatic properties is difficult because studies are often conducted under disparate experimental conditions and techniques. Here, we directly compare and reveal functional differences between Saccharomyces cerevisiae Pols I and II using a series of quantitative in vitro transcription assays. We find that Pol I single-nucleotide and multinucleotide addition rate constants are faster than those of Pol II. Pol I elongation complexes are less stable than Pol II elongation complexes, and Pol I is more error prone than Pol II. Collectively, these data show that the enzymatic properties of the Pols have diverged over the course of evolution, optimizing these enzymes for their unique cellular responsibilities.
Assuntos
RNA Polimerase II/metabolismo , RNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Cinética , Polimorfismo de Nucleotídeo Único , Transcrição GênicaRESUMO
Complementary and alternative medicine (CAM) includes a wide range of practices and products that are generally outside the use of conventional medicine as practiced in Western cultures. Use of CAM in persons with epilepsy is high, even compared to individuals with other chronic health conditions. In this study, we surveyed caregivers of children admitted to a regional epilepsy monitoring unit (EMU) in the southeast United States to assess CAM use among patients (N=225). Thirteen percent of respondents indicated current use of CAM by their child, 16% reported past use, and 43% reported interest in future use, most commonly in marijuana as a potential treatment (23%). Over 25% of respondents expressed interest in CAM use related to side effects of anti-epileptic medications. Regarding prayer as a form of CAM, a large majority of respondents in this sample identified as Christian and actively prayed for their child's illness, revealing a high prevalence of spiritual practices in this population. Eighty-one percent of respondents reported that they had not discussed CAM use with their doctor. Discussing CAM use with a health care provider was significantly related to past CAM use (p<.02), but not current use or willingness to try CAM in the future (p>.05). These results have important implications for future practice and support increased communication and patient education, as many anti-epileptic medications interact with certain herbs and supplements, posing a potential health risk and treatment barrier in this population.
Assuntos
Cuidadores/psicologia , Terapias Complementares/psicologia , Epilepsia/psicologia , Monitorização Fisiológica/tendências , Admissão do Paciente/tendências , Religião , Adolescente , Adulto , Anticonvulsivantes/uso terapêutico , Criança , Pré-Escolar , Terapias Complementares/métodos , Estudos Transversais , Epilepsia/epidemiologia , Epilepsia/terapia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Monitorização Fisiológica/métodos , Prevalência , Inquéritos e Questionários , Adulto JovemRESUMO
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.
Assuntos
Difosfatos , RNA Polimerase II , Saccharomyces cerevisiae , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Cinética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Difosfatos/metabolismo , Nucleotídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
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.
Assuntos
RNA Polimerase I , Cinética , RNA Polimerase I/metabolismo , RNA Polimerase I/química , Nucleotídeos/metabolismo , Nucleotídeos/químicaRESUMO
Here we present an optimized protocol for monitoring and analyzing single nucleotide incorporation by RNA polymerases. This protocol describes the assembly of Saccharomyces cerevisiae RNA polymerase I elongation complexes in a promoter-independent system in vitro. We describe how to collect a time course using a quench-flow, a rapid mixing instrument, and subsequently resolve reactions on a polyacrylamide gel. Finally, we detail how to quantify the gel images. For complete details on the use and execution of this protocol, please refer to Appling et al. (2015).1.
RESUMO
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.
Assuntos
Difosfatos , RNA Polimerase I , Difosfatos/metabolismo , Exonucleases , Nucleotídeos/metabolismo , RNA Polimerase I/química , RNA Polimerase I/genética , RNA Polimerase I/metabolismoRESUMO
Eukaryotes express three nuclear RNA polymerases (Pols I, II, and III) that are essential for cell survival. Despite extensive investigation of the three Pols, significant knowledge gaps regarding their biochemical properties remain because each Pol has been evaluated independently under disparate experimental conditions and methodologies. To advance our understanding of the Pols, we employed identical in vitro transcription assays for direct comparison of their elongation rates, elongation complex (EC) stabilities, and fidelities. Pol I is the fastest, most likely to misincorporate, forms the least stable EC, and is most sensitive to alterations in reaction buffers. Pol II is the slowest of the Pols, forms the most stable EC, and negligibly misincorporated an incorrect nucleotide. The enzymatic properties of Pol III were intermediate between Pols I and II in all assays examined. These results reveal unique enzymatic characteristics of the Pols that provide new insights into their evolutionary divergence.
RESUMO
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.