Investigation of the catalytic mechanism of RNase P: the role of divalent metal ions and functional groups important for catalysis

The ribonucleoprotein enzyme ribonuclease (RNase) P is an endonuclease that generates the mature 5' -ends of tRNA. Bacterial RNase P is composed of a large RNA subunit (P RNA) and a small protein (P protein). Studies with P RNA from E. coli and B. subtilis have implied a specific role for two or more metal ions in substrate binding and cleavage chemistry. In this study it is demonstrated, for the first time, catalysis by E. coli P RNA with zinc as the sole divalent metal ion cofactor. Although proficient in catalysis, zinc destabilises E•S complex formation. In contrast, strontium inhibits catalysis, but promotes high substrate affinity. Stimulating and inhibitory effects of strontium could be rationalised by a model involving two strontium ions (or two classes), both improving substrate affinity in a cooperative manner, but one of the two inhibiting substrate conversion in a noncompetitive mode with respect to substrate. Further analyses suggest that the inhibition mode of strontium is noncompetitive with respect to zinc. The 2'-OH group at the scissile phosphodiester (nt –1 of ptRNA) contributes to positioning of a catalytic metal ion and may donate a H-bond to the 3'-O leaving group. NMR analyses have indicated that the ribose at nt –1 predominantly populates the C2'-endo conformation. Since the energy barrier for interconversion of C2'- and C3'-endo puckering is expected to be low, it is unknown which conformation is adopted during P RNA catalysis. To address this issue, we analysed cleavage of a ptRNA carrying an locked nucleic acid (LNA) substitution at nt –1, LNA being the only well known substituent that locks the ribose in a C3'-endo puckering. A 2'-methoxy substitution at this position was analysed in parallel, since it is chemically closely related to LNA. Other variants with 2'-fluoro or 2'-deoxy substitution at nt –1 were included in this study. An LNA substitution at nt –1 dramatically reduced (more that a 2'-deoxy) cleavage at the canonical site (-1/+1), while the effects on ground state binding were marginal. A 2'-methoxy substitution completely abolished cleavage at the canonical site. Also, both substituents suppressed cleavage at the site -1/-2. Instead, aberrant cleavage at the site +1/+2 was observed. Since the cleavage at the canonical site of the substrate with LNA at nt –1 had a higher magnesium requirement compared to cleavage at the +1/+2 site, it is likely that the methylene group of LNA inhibit cleavage at the canonical site by sterical interference with a catalytic magnesium ion. The fact that LNA at nt –1 still permitted residual cleavage at the canonical site indicates that the transition state can be reached in the presence of a locked C3'-endo conformation at nt –1. We further tested LNA at nt +1. Here, LNA had only a marginal effect on cleavage chemistry, but significantly reduced ptRNA binding affinity. The binding defect could be overcome at high metal ion concentrations. Again, comparison with a 2'-methoxy and 2'-deoxy modification indicated sterical hindrance by the methylen or methyl group. Hill analysis of metal ion dependence of ptRNA binding revealed a higher metal ion cooperativity for the LNA variant compared to the unmodified one, indicating that the methylene group sterically interferes, directly or indirectly, with metal ion binding to at least one site crucial for E•S complex formation. Functional groups within the P RNA and/or ptRNA are essential for substrate binding and cleavage and they can be mapped by modification interference studies: nucleotide analogue interference mapping (NAIM) and suppression (NAIS). For this purpose, an RNA chimera consisting of E. coli P RNA and the tRNA 5'-half was constructed. A functional substrate was reconstituted by annealing the tRNA 5'-half with its 3'-half resulting in a cis-cleaving RNA complex. A partially modified RNA pool (RNA chimera) was then synthesised by in vitro transcription. After separation of functional (cis-cleaving) and non-functional molecules, positions within the 3' -half of the P RNA where modifications interfered with processing (cis-cleavage reaction) could be identified. These positions are overlapping to some extent with those found in a previous study, where interference with E. coli P RNA - tRNA binding was analysed. These results are to be expected because functional groups in P RNA important for substrate binding are likewise important for substrate turnover, since binding is a prerequisite for cleavage. A strong interference effect at G 350 was detected only with the cis – cleavage assay. G 350 may represent a position essential for the catalytic step, as evidence was provided that it contributes to the binding of catalytically important magnesium near the active site of P RNA

Studies on Escherichia coli RNase P RNA with Zn(2+) as the catalytic cofactor

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn(2+) as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn(2+) ions in catalysis. Whereas Zn(2+) destabilizes substrate ground state binding to an extent that precludes reliable K(d) determination, [Formula: see text] and Sr(2+) in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn(2+) and [Formula: see text] substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr(2+) on the ribozyme reaction with Zn(2+) as cofactor could be rationalized by a model involving two Sr(2+) ions (or two classes of Sr(2+) ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn(2+). A single 2′-fluoro modification at nt −1 of the substrate substantially weakened the inhibitory effect of Sr(2+). Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg(2+) entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E·S formation apart from the catalytic performance

Investigation of the catalytic mechanism of RNase P: the role of divalent metal ions and functional groups important for catalysis

The ribonucleoprotein enzyme ribonuclease (RNase) P is an endonuclease that generates the mature 5' -ends of tRNA. Bacterial RNase P is composed of a large RNA subunit (P RNA) and a small protein (P protein). Studies with P RNA from E. coli and B. subtilis have implied a specific role for two or more metal ions in substrate binding and cleavage chemistry. In this study it is demonstrated, for the first time, catalysis by E. coli P RNA with zinc as the sole divalent metal ion cofactor. Although proficient in catalysis, zinc destabilises E•S complex formation. In contrast, strontium inhibits catalysis, but promotes high substrate affinity. Stimulating and inhibitory effects of strontium could be rationalised by a model involving two strontium ions (or two classes), both improving substrate affinity in a cooperative manner, but one of the two inhibiting substrate conversion in a noncompetitive mode with respect to substrate. Further analyses suggest that the inhibition mode of strontium is noncompetitive with respect to zinc. The 2'-OH group at the scissile phosphodiester (nt –1 of ptRNA) contributes to positioning of a catalytic metal ion and may donate a H-bond to the 3'-O leaving group. NMR analyses have indicated that the ribose at nt –1 predominantly populates the C2'-endo conformation. Since the energy barrier for interconversion of C2'- and C3'-endo puckering is expected to be low, it is unknown which conformation is adopted during P RNA catalysis. To address this issue, we analysed cleavage of a ptRNA carrying an locked nucleic acid (LNA) substitution at nt –1, LNA being the only well known substituent that locks the ribose in a C3'-endo puckering. A 2'-methoxy substitution at this position was analysed in parallel, since it is chemically closely related to LNA. Other variants with 2'-fluoro or 2'-deoxy substitution at nt –1 were included in this study. An LNA substitution at nt –1 dramatically reduced (more that a 2'-deoxy) cleavage at the canonical site (-1/+1), while the effects on ground state binding were marginal. A 2'-methoxy substitution completely abolished cleavage at the canonical site. Also, both substituents suppressed cleavage at the site -1/-2. Instead, aberrant cleavage at the site +1/+2 was observed. Since the cleavage at the canonical site of the substrate with LNA at nt –1 had a higher magnesium requirement compared to cleavage at the +1/+2 site, it is likely that the methylene group of LNA inhibit cleavage at the canonical site by sterical interference with a catalytic magnesium ion. The fact that LNA at nt –1 still permitted residual cleavage at the canonical site indicates that the transition state can be reached in the presence of a locked C3'-endo conformation at nt –1. We further tested LNA at nt +1. Here, LNA had only a marginal effect on cleavage chemistry, but significantly reduced ptRNA binding affinity. The binding defect could be overcome at high metal ion concentrations. Again, comparison with a 2'-methoxy and 2'-deoxy modification indicated sterical hindrance by the methylen or methyl group. Hill analysis of metal ion dependence of ptRNA binding revealed a higher metal ion cooperativity for the LNA variant compared to the unmodified one, indicating that the methylene group sterically interferes, directly or indirectly, with metal ion binding to at least one site crucial for E•S complex formation. Functional groups within the P RNA and/or ptRNA are essential for substrate binding and cleavage and they can be mapped by modification interference studies: nucleotide analogue interference mapping (NAIM) and suppression (NAIS). For this purpose, an RNA chimera consisting of E. coli P RNA and the tRNA 5'-half was constructed. A functional substrate was reconstituted by annealing the tRNA 5'-half with its 3'-half resulting in a cis-cleaving RNA complex. A partially modified RNA pool (RNA chimera) was then synthesised by in vitro transcription. After separation of functional (cis-cleaving) and non-functional molecules, positions within the 3' -half of the P RNA where modifications interfered with processing (cis-cleavage reaction) could be identified. These positions are overlapping to some extent with those found in a previous study, where interference with E. coli P RNA - tRNA binding was analysed. These results are to be expected because functional groups in P RNA important for substrate binding are likewise important for substrate turnover, since binding is a prerequisite for cleavage. A strong interference effect at G 350 was detected only with the cis – cleavage assay. G 350 may represent a position essential for the catalytic step, as evidence was provided that it contributes to the binding of catalytically important magnesium near the active site of P RNA

Riscuri de dezvoltare a bolilor profesionale (sindromul burnout)

Introduction. The medical profession is one of the noblest and most necessary professions in the world, but at the same time one of the most difficult. Psychological training of students plays an important role in the development of a doctor as a specialist. Objective of the study. The proposed goal is to analyze the syndrome of “emotional (or mental) exhaustion” which is a state of physical, emotional and mental exhaustion, negative attitude towards work, loss of empathy, compassion and understanding in relation to patients and their loved ones. Medical workers have the highest burnout occupations. Above all, those who make unreasonably high demands are at risk of developing Burnout. Materials and methods. The investigative methods used are theoretical and praxiological: scientific documentation, observation, and analysis of the investigated material. Results. The results of the study show that the increased workload, overtime hours stimulate the development of burnout. Breaks at work have a positive effect and reduce exhaustion, but this effect is temporary. Doctors get rid of this situation by using psychotropic substances. Conclusion. When it comes to preventing exhaustion in the medical profession, each of us should be encouraged to learn how to reprioritize and think about lifestyle changes. It is necessary to be able to plan our activities properly, to respect the work and rest schedule. It is also necessary to actively apply methods of psychological relief and reduction of emotional tension.Introducere. Profesia de medic este una dintre cele mai nobile și necesare meserii din lume, dar în același timp una dintre cele mai dificile. Pregătirea psihologică a studenților joacă un rol important în dezvoltarea unui medic ca specialist. Scopul lucrării. Scopul propus vizează analiza sindromului de „epuizare emoțională (sau mentală)” care este o stare de epuizare fizică, emoțională și mentală, atitudinea negativă față de muncă, pierderea empatiei, a compasiunii și a înțelegerii în relație cu pacienții și cei dragi lor. Lucrătorii medicali au profesii cu cea mai mare tendință de „burnout”. Mai presus de toate, cei care își fac cerințe nerezonabil de mari sunt expuși riscului de a dezvolta sindromul Burnout. Materiale și metode. Metodele de investigație utilizate sunt teoretice și praxiologice: documentarea științifică, observația și analiza materialului investigat. Rezultate. Rezultatele studiului constată că volumul de muncă crescut, orele de lucru suplimentare stimulează dezvoltarea burnout-ului. Pauzele la lucru au un efect pozitiv și reduc epuizarea, dar acest efect este temporar. Medicii scapă de situația aceasta prin utilizarea de substanțe psihotrope. Concluzii. Vorbind despre prevenirea epuizării în profesia medicală, fiecare dintre noi ar trebui încurajat să învețe cum să reprioritizeze și să se gândească la schimbările stilului de viață. Este necesar să ne putem planifica în mod corespunzător activitățile, să respectam programul de muncă și de odihnă. De asemenea, este necesar să se aplice în mod activ metodele de ameliorare psihologică și de reducere a tensiunii emoționale

RISKS OF THE DEVELOPMENT OF OCCUPATIONAL DISEASES (BURNOUT SYNDROME)

Universitatea de Stat de Medicină şi Farmacie „Nicolae Testemiţanu”, Chişinău, Republica MoldovaIntroducere. Profesia de medic este una dintre cele mai nobile și necesare meserii din lume, dar în același timp una dintre cele mai dificile. Pregătirea psihologică a studenților joacă un rol important în dezvoltarea unui medic ca specialist. Scopul lucrării. Scopul propus vizează analiza sindromului de „epuizare emoțională (sau mentală)” care este o stare de epuizare fizică, emoțională și mentală, atitudinea negativă față de muncă, pierderea empatiei, a compasiunii și a înțelegerii în relație cu pacienții și cei dragi lor. Lucrătorii medicali au profesii cu cea mai mare tendință de „burnout”. Mai presus de toate, cei care își fac cerințe nerezonabil de mari sunt expuși riscului de a dezvolta sindromul Burnout. Materiale și metode. Metodele de investigație utilizate sunt teoretice și praxiologice: documentarea științifică, observația și analiza materialului investigat. Rezultate. Rezultatele studiului constată că volumul de muncă crescut, orele de lucru suplimentare stimulează dezvoltarea burnout-ului. Pauzele la lucru au un efect pozitiv și reduc epuizarea, dar acest efect este temporar. Medicii scapă de situația aceasta prin utilizarea de substanțe psihotrope. Concluzii. Vorbind despre prevenirea epuizării în profesia medicală, fiecare dintre noi ar trebui încurajat să învețe cum să reprioritizeze și să se gândească la schimbările stilului de viață. Este necesar să ne putem planifica în mod corespunzător activitățile, să respectam programul de muncă și de odihnă. De asemenea, este necesar să se aplice în mod activ metodele de ameliorare psihologică și de reducere a tensiunii emoționale.Introduction. The medical profession is one of the noblest and most necessary professions in the world, but at the same time one of the most difficult. Psychological training of students plays an important role in the development of a doctor as a specialist. Objective of the study. The proposed goal is to analyze the syndrome of “emotional (or mental) exhaustion” which is a state of physical, emotional and mental exhaustion, negative attitude towards work, loss of empathy, compassion and understanding in relation to patients and their loved ones. Medical workers have the highest burnout occupations. Above all, those who make unreasonably high demands are at risk of developing Burnout. Materials and methods. The investigative methods used are theoretical and praxiological: scientific documentation, observation, and analysis of the investigated material. Results. The results of the study show that the increased workload, overtime hours stimulate the development of burnout. Breaks at work have a positive effect and reduce exhaustion, but this effect is temporary. Doctors get rid of this situation by using psychotropic substances. Conclusion. When it comes to preventing exhaustion in the medical profession, each of us should be encouraged to learn how to reprioritize and think about lifestyle changes. It is necessary to be able to plan our activities properly, to respect the work and rest schedule. It is also necessary to actively apply methods of psychological relief and reduction of emotional tension

Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester

We analyzed cleavage of precursor tRNAs with an LNA, 2′-OCH3, 2′-H or 2′-F modification at the canonical (c0) site by bacterial RNase P. We infer that the major function of the 2′-substituent at nt −1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c0 site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3′ -endo ribose and without the H-bond donor function of the 2′-substituent. LNA and 2′-OCH3 suppressed processing at the major aberrant m−1 site; instead, the m+1 (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c0 and m+1 sites had different pH profiles, with a higher Mg2+ requirement for c0 versus m+1 cleavage. The strong catalytic defect for LNA and 2′-OCH3 supports a model where the extra methylene (LNA) or methyl group (2′-OCH3) causes a steric interference with a nearby bound catalytic Mg2+ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects

Minor changes largely restore catalytic activity of archaeal RNase P RNA from Methanothermobacter thermoautotrophicus

The increased protein proportion of archaeal and eukaryal ribonuclease (RNase) P holoenzymes parallels a vast decrease in the catalytic activity of their RNA subunits (P RNAs) alone. We show that a few mutations toward the bacterial P RNA consensus substantially activate the catalytic (C-) domain of archaeal P RNA from Methanothermobacter, in the absence and presence of the bacterial RNase P protein. Large increases in ribozyme activity required the cooperative effect of at least two structural alterations. The P1 helix of P RNA from Methanothermobacter was found to be extended, which increases ribozyme activity (ca 200-fold) and stabilizes the tertiary structure. Activity increases of mutated archaeal C-domain variants were more pronounced in the context of chimeric P RNAs carrying the bacterial specificity (S-) domain of Escherichia coli instead of the archaeal S-domain. This could be explained by the loss of the archaeal S-domain's capacity to support tight and productive substrate binding in the absence of protein cofactors. Our results demonstrate that the catalytic capacity of archaeal P RNAs is close to that of their bacterial counterparts, but is masked by minor changes in the C-domain and, particularly, by poor function of the archaeal S-domain in the absence of archaeal protein cofactors

The putative RNase P motif in the DEAD box helicase Hera is dispensable for efficient interaction with RNA and helicase activity

DEAD box helicases use the energy of ATP hydrolysis to remodel RNA structures or RNA/protein complexes. They share a common helicase core with conserved signature motifs, and additional domains may confer substrate specificity. Identification of a specific substrate is crucial towards understanding the physiological role of a helicase. RNA binding and ATPase stimulation are necessary, but not sufficient criteria for a bona fide helicase substrate. Here, we report single molecule FRET experiments that identify fragments of the 23S rRNA comprising hairpin 92 and RNase P RNA as substrates for the Thermus thermophilus DEAD box helicase Hera. Both substrates induce a switch to the closed conformation of the helicase core and stimulate the intrinsic ATPase activity of Hera. Binding of these RNAs is mediated by the Hera C-terminal domain, but does not require a previously proposed putative RNase P motif within this domain. ATP-dependent unwinding of a short helix adjacent to hairpin 92 in the ribosomal RNA suggests a specific role for Hera in ribosome assembly, analogously to the Escherichia coli and Bacillus subtilis helicases DbpA and YxiN. In addition, the specificity of Hera for RNase P RNA may be required for RNase P RNA folding or RNase P assembly

Assessment of Different Techniques for Adhesive Cementation of All-Ceramic Systems

Background and Objectives: Modern esthetic dentistry is based on all-ceramic restorations. Dentists still have reservations about using these restorations due to a lack of understanding of the cementation technique, which depends on the type of ceramic used. The aim of the study is to evaluate the approaches and practices of clinicians regarding the adhesive cementation of all-ceramic restorations. Materials and Methods: An online questionnaire regarding the use of all-ceramic restorations and their bonding methods was designed by distinguishing the cementation of oxide and silica-based ceramics. The survey included dentists practicing in Timiș County, Romania. The questionnaire and the evaluation of the answers were designed based on the techniques and evidence from the literature. Results: Considering the work experience, we obtained two groups: group 1—1 to 6 years and group 2—6 to 9+ years. The results revealed significant values when comparing the two groups in the surface protocol and decontamination (p = 0.005), type of cement used (p = 0.002), and isolation techniques (p = 0.002). Conclusions: The results show that many clinicians need additional training to improve their cementing technique and avoid the confusion caused by insufficient information about the interrelationship between the type of ceramic and the cementation procedure