RESUMO
The chemical nature of the 5' end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency, and has been proposed to provide a layer of 'epitranscriptomic' gene regulation. Recently it has been shown that some bacterial RNA species carry a 5'-end structure reminiscent of the 5' 7-methylguanylate 'cap' in eukaryotic RNA. In particular, RNA species containing a 5'-end nicotinamide adenine dinucleotide (NAD+) or 3'-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated 'ab initio capping' may occur in all organisms.
Assuntos
Coenzima A/metabolismo , NAD/metabolismo , Capuzes de RNA/metabolismo , Iniciação da Transcrição Genética , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Dados de Sequência Molecular , Nucleotídeos/química , Nucleotídeos/metabolismo , Regiões Promotoras Genéticas/genética , Capuzes de RNA/química , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
A rapid gravimetric method for determining drug candidate solubility in organic solvents has been developed. The scale, speed, precision, and accuracy of the method make it ideal for solubility screening of pharmaceutical compounds during early development. The method utilizes a thermogravimetric analyzer to automate drying and weighing. Results for model compounds compare favorably with literature values.
Assuntos
Compostos Orgânicos/química , Solventes/química , Reprodutibilidade dos Testes , SolubilidadeRESUMO
Modulated thermogravimetic analysis (MTGA) is evaluated for the rapid estimation of thermal stability using several penicillin antibiotics as model compounds. The MTGA technique utilizes an oscillatory temperature program to obtain Arrhenius kinetic parameters through a mass loss during thermal degradation. To evaluate the reliability of this technique, activation energies (E(a)), log pre-exponential factor (logZ), and log first order rate constants (logk) obtained by MTGA for the thermal decomposition of ampicillin anhydrous, ampicillin trihydrate, ampicillin sodium salt, and penicillin G potassium salt are compared to existing literature values. The logk values estimated by MTGA agreed well with literature values when the weight loss observed by MTGA was shown to be due to the first decomposition step of the compound. The E(a) and logZ values determined by MTGA did not consistently agree with literature values as these parameters increased with decreasing heating rate (beta). The increase in E(a) and logZ values with decreasing beta seemed to offset each other to some extent to yield a relatively consistent logk estimate regardless of beta.
Assuntos
Temperatura Alta , Penicilinas/análise , Varredura Diferencial de Calorimetria/métodos , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Cinética , Modelos Químicos , Fatores de TempoRESUMO
The thermodynamically most stable polymorph under ambient conditions is almost without exception the most desirable crystalline form for development by a pharmaceutical company. It is, therefore, beneficial to discover and to characterize this polymorph at the earliest possible stage of development. A screen for discovering the stable polymorph of a pharmaceutical compound early in the drug discovery-development process is developed and described. In this screen, a small amount of compound is suspended in a diverse group of solvents for two weeks in an effort to crystallize the most stable polymorph. The solubility of the compound in each solvent utilized in the stable polymorph screen is also simultaneously determined using a simple gravimetric method. Ritonavir and an early development candidate (Pfizer compound A) are used as model compounds to demonstrate the utility of the screen for finding the stable polymorph early in the drug discovery-development process.