Background and Purpose Hydrogen sulfide ( H 2 S ) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H 2 S are cystathionine β synthase ( CBS ) and cystathionine γ lyase ( CSE ). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H 2 S biosynthesis towards CSE and CBS . Experimental Approach To address this question, human CSE or CBS enzymes were expressed and purified from E scherichia coli as fusion proteins with GSH‐ S ‐transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method. Key Results β‐cyanoalanine ( BCA ) was more potent in inhibiting CSE than propargylglycine ( PAG ) ( IC 50 14 ± 0.2 μM vs. 40 ± 8 μM respectively). Similar to PAG , L ‐aminoethoxyvinylglycine ( AVG ) only inhibited CSE , but did so at much lower concentrations. On the other hand, aminooxyacetic acid ( AOAA ), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG ( IC 50 1.1 ± 0.1 μM); the IC 50 for AOAA for inhibiting CBS was 8.5 ± 0.7 μM. In line with our biochemical observations, relaxation to L‐cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H 2 S biosynthesis, blocked the activity of CBS and CSE . Trifluoroalanine had a fourfold lower IC 50 for CBS versus CSE , while hydroxylamine was 60‐fold more selective against CSE . Conclusions and Implications In conclusion, although PAG , AVG and BCA exhibit selectivity in inhibiting CSE versus CBS , no selective pharmacological CBS inhibitor is currently available.
Indirect evidence has abundantly been presented to support the view that substance P (SP) is involved in the vasodilatation following activation of fine calibre pain fibres (Lembeck & Holzer 1979). In this respect, the dental pulp is interesting since it is richly supplied with SP‐immunoreactive nerves originating from the trigeminal system (Olgart et al. 1917 b , Brodin et al. 1980). These nerves are in all probability related to nociception (Henry et al. 1980). Recent observations in the cat showing that electrical stimulation of the inferior alveolar nerve produces an atropine resistant vasodilatation (Gazelius & Olgart 1980), and a release of substance P‐like immunoreactivity in the pulp (Olgart et al. 1977a, Brodin et al. 1980) are in accord with the suggested role of SP. In the present study, the availability of an antagonist to SP has made possible the further elucidation of the role of SP as a mediator of antidromic vasodilatation in the dental pulp and in the oral mucosa of the cat. We have also determined whether (D‐Pro 2 , D‐Phe 7 , D‐Trp 9 )‐SP specifically blocks the vascular effects of SP.
Yeast ribosomal protein L41 is dispensable in the yeast. Its absence had no effect on polyphenylalanine synthesis activity, and a limited effect on growth, translational accuracy, or the resistance toward the antibiotic paromomycin. Removal of L41 did not affect the 60:40 S ratio, but it reduced the amount of 80 S, suggesting that L41 is involved in ribosomal subunit association. However, the two most important effects of L41 were on peptidyltransferase activity and translocation. Peptidyltransferase activity was measured as a second-order rate constant (k cat/K s) corresponding to the rate of peptide bond formation; thisk cat/K s was lowered 3-fold to 1.15 min−1 mm−1 in the L41 mutant compared with 3.46 min−1mm−1 in the wild type. Translocation was also affected by L41. Elongation factor 2 (EF2)-dependent (enzymatic) translocation of Ac-Phe-tRNA from the A- to P-site was more efficient in the absence of L41, because 50% translocation was achieved at only 0.004 μm EF2 compared with 0.02 μm for the wild type. Furthermore, the EF2-dependent translocation was inhibited by 50% at 2.5 μm of the translocation inhibitor cycloheximide in the L41 mutant compared with 1.2 μm in the wild type. Finally, the rate of EF2-independent (spontaneous) translocation was increased in the absence of L41. Yeast ribosomal protein L41 is dispensable in the yeast. Its absence had no effect on polyphenylalanine synthesis activity, and a limited effect on growth, translational accuracy, or the resistance toward the antibiotic paromomycin. Removal of L41 did not affect the 60:40 S ratio, but it reduced the amount of 80 S, suggesting that L41 is involved in ribosomal subunit association. However, the two most important effects of L41 were on peptidyltransferase activity and translocation. Peptidyltransferase activity was measured as a second-order rate constant (k cat/K s) corresponding to the rate of peptide bond formation; thisk cat/K s was lowered 3-fold to 1.15 min−1 mm−1 in the L41 mutant compared with 3.46 min−1mm−1 in the wild type. Translocation was also affected by L41. Elongation factor 2 (EF2)-dependent (enzymatic) translocation of Ac-Phe-tRNA from the A- to P-site was more efficient in the absence of L41, because 50% translocation was achieved at only 0.004 μm EF2 compared with 0.02 μm for the wild type. Furthermore, the EF2-dependent translocation was inhibited by 50% at 2.5 μm of the translocation inhibitor cycloheximide in the L41 mutant compared with 1.2 μm in the wild type. Finally, the rate of EF2-independent (spontaneous) translocation was increased in the absence of L41. acetyl elongation factor 2 soluble protein factors The ribosome is a large macromolecular machine that consists of a large number of proteins and several molecules of ribosomal RNA (rRNA). The ribosome is responsible for the translation of the genetic message, which results in protein synthesis.The crystal structures of the 30 S (1Wimberly B.T. Brodersen D.E. Clemons W.M. Morgan-Warren R.J. Carter A.P. Vonrhein C. Hartsch T. Ramakrishnan V. Nature. 2000; 407: 327-348Google Scholar, 2Schluenzen F. Tocilj A. Zarivach R. Harms J. Gluehmann M. Janell D. Bashan A. Bartels H. Agmon I. Franceschi F. Yonath A. Cell. 2000; 102: 615-623Google Scholar) and 50 S (3Ban N. Nissen P. Hansen J. Moore P.B. Steitz T.A. Science. 2000; 289: 905-920Google Scholar, 4Harms J. Schluenzen F. Zarivach R. Bashan A. Gat S. Agmon I. Bartels H. Franceschi F. Yonath A. Cell. 2001; 107: 679-688Google Scholar) ribosomal subunits, and the intact 70 S ribosome (5Yusupov M.M. Yusupova G.Z. Baucom A. Lieberman K. Earnest T.N. Cate J.H. Noller H.F. Science. 2001; 292: 883-896Google Scholar) are contributing to a better understanding of ribosomal function. It has now been confirmed that rRNA plays the major role in ribosomal structure and function, including the two most important activities of the ribosome: the decoding process and the peptidyltransferase activity. The 16 S rRNA of the small ribosomal subunit is responsible for the decoding process, the selection of the cognate tRNA (6Carter A.P. Clemons W.M., Jr. Brodersen D.E. Morgan-Warren R.J. Wimberly B.T. Ramakrishnan V. Nature. 2000; 407: 340-348Google Scholar). The central loop of domain V of 23 S rRNA of the large ribosomal subunit is responsible for the catalytic activity of the ribosome (7Nissen P. Hansen J. Ban N. Moore P.B. Steitz T.A. Science. 2000; 289: 920-930Google Scholar, 8Muth G.W. Ortoleva–Donnelly L. Strobel S.A. Science. 2000; 289: 947-950Google Scholar). The search is still underway to identify a critical site or nucleotide(s) of 23 S rRNA involved in the catalysis of peptide bond formation (9O'Connor M. Lee W-C.M. Mankad A. Squires C.L. Dahlberg A.E. Nucleic Acids Res. 2001; 29: 710-715Google Scholar, 10Bayfield M.A. Dahlberg E.A. Schulmeister U. Dorner S. Barta A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10096-10101Google Scholar, 11Xiong L. Polacek N. Sander P. Böttger E.C. Mankin A. RNA. 2001; 7: 1365-1369Google Scholar, 12Thompson J. Kim D.F. O'Connor M. Lieberman K.R. Bayfield M.A. Gregory S.T. Green R. Noller H.F. Dahlberg A.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9002-9007Google Scholar, 13Schmeing T.M. Seila A.C. Hansen J.L. Freeborn B. Soukup J.K. Scaringe S.A. Strobel S.A. Moore P.B. Steitz T.A. Nat. Struct. Biol. 2002; 9: 225-230Google Scholar). It is still possible that the rate of peptide bond formation is influenced by structural rearrangements of RNA in the peptidyltransferase center that would affect the positioning of the substrates (14Polacek N. Gaynor M. Yassin A. Mankin A.S. Nature. 2001; 411: 498-501Google Scholar).Ribosomal proteins offer structural support to the ribosome by stabilizing and orienting the ribosomal RNA into a specific, active structure (15Cech T.R. Science. 2000; 289: 878-879Google Scholar). Also, they are crucial for the assembly of functional ribosomes (16Stern S. Powers T. Changchien L.M. Noller H.F. Science. 1989; 244: 783-790Google Scholar). Several ribosomal proteins of the small subunit (17Vincent A. Liebman S.W. Genetics. 1992; 132: 375-386Google Scholar, 18Alksne L.E. Anthony R.A. Liebman S.W. Warner J.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9538-9541Google Scholar, 19Liebman S.W. Chernoff Y.O. Liu R. Biochem. Cell Biol. 1995; 73: 1141-1149Google Scholar, 20Synetos D. Frantziou C.P. Alksne L.E. Biochim. Biophys. Acta. 1996; 1309: 156-166Google Scholar) and at least one of the large subunit (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar) appear to affect the decoding process. Moreover, some ribosomal proteins appear to influence the peptidyltransferase activity of the ribosome (22Wower I.K. Wower J. Zimmermann R.A. J. Biol. Chem. 1998; 273: 19847-19852Google Scholar, 23Diedrich G. Spahn C.M.T. Stelzl U. Schäfer M.A. Wooten T. Bochkariov D.E. Cooperman B.S. Traut R.R. Nierhaus K.H. EMBO J. 2000; 19: 5241-5250Google Scholar, 24Dresios J. Panopoulos P. Frantziou C.P. Synetos D. Biochemistry. 2001; 40: 8101-8108Google Scholar).Yeast ribosomal protein L41 is the smallest and the most basic eukaryotic protein. Its open reading frame is composed of only 25 amino acids, 17 of which are arginines or lysines (25Suzuki K. Hashimoto T. Otaka E. Curr. Genet. 1990; 17: 185-190Google Scholar). L41 is highly conserved in eukaryotes and it is present in certain archaea,e.g. Methanococcus jannaschii, but not in eubacteria. A protein with similar properties is present in certain thermophilic eubacteria, e.g. Thermus thermophilus (26Choli T. Franceschi F. Wittmann-Liebold B. Yonath A. Biol. Chem. Hoppe-Seyler. 1993; 374: 377-383Google Scholar). In Saccharomyces cerevisiae, L41 is encoded by two genes, RPL41A and RPL41B (25Suzuki K. Hashimoto T. Otaka E. Curr. Genet. 1990; 17: 185-190Google Scholar). In a recent study, the properties and the translation of the mRNAs encoding L41 were investigated (27Yu X. Warner J.R. J. Biol. Chem. 2001; 276: 33821-33825Google Scholar). It was found that the L41 mRNAs are translated exclusively on monosomes and the entire translation process from initiation through to termination occurs in about 2 s. However, as with most ribosomal proteins, the function of L41 remains unknown (27Yu X. Warner J.R. J. Biol. Chem. 2001; 276: 33821-33825Google Scholar).There is evidence that suggests that protein L41 may possess extraribosomal activity; it associates directly in vitrowith subunit b of protein kinase CKII, although it is not a substrate for CKII phosphorylation. However, L41 protein stimulates the phosphorylation of DNA topoisomerase IIa by CKII. Additionally, L41 enhances the autophosphorylation of CKIIa (28Lee J.H. Kim J.M. Lee Y.T. Marshak D.R. Bae Y.S. Biochem. Biophys. Res. Commun. 1997; 238: 462-467Google Scholar). These data indicate that L41 associates with CKII and can modulate its activity toward a specific substrate(s).In the present work we tried to gain insight into the role of protein L41 in protein synthesis. For this purpose we examined two yeast strains. The first lacked the two genes encoding L41. The second strain lacked L41 in addition to the absence of L24 and L39, two eukaryote-specific proteins studied recently (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar, 24Dresios J. Panopoulos P. Frantziou C.P. Synetos D. Biochemistry. 2001; 40: 8101-8108Google Scholar). The fact that the cells remained viable upon removal of the two genes encoding L41 in addition to the absence of the two genes encoding L24 and the single gene encoding L39, allowed for the first time the study of a quintuple mutant in yeast as well as the study of the role of L41 in combination with the effects of the other two proteins. Our results suggest that a dispensable ribosomal protein such as L41 may affect in varying degrees several important functions of yeast ribosomes confirming the interconnectedness of these ribosomal activities.DISCUSSIONIn an effort to contribute to the understanding of yeast ribosomal structure and function, we examined the role(s) of ribosomal protein L41. For this purpose we used two strains, one in which the two genes for L41 were deleted and a second strain, in which the two genes for L41 were deleted from a background already lacking the genes for L24 and L39. We have found that in the absence of L24 and L39 the cell exhibited reduced protein synthesis activity and decreased translational accuracy, two closely related functions (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar). Thus, this was an appropriate background in which to study the effect of the absence of a third protein, L41, on the above functions.We found that protein L41 is dispensable for the yeast S. cerevisiae. This is the third case of a eukaryotic ribosomal protein, not found in eubacteria, which is nonessential for cell viability. The other two nonessential proteins, L24 and L39, were found to have significant effects on certain parameters of protein synthesis (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar, 24Dresios J. Panopoulos P. Frantziou C.P. Synetos D. Biochemistry. 2001; 40: 8101-8108Google Scholar). Specifically, L24 acted on the assembly of ribosomes and the kinetics of protein synthesis, whereas L39 acted on the accuracy of translation and the assembly of the 60 S subunits. Protein L41 is dispensable but it is not entirely innocuous to the cell: doubling times were somewhat longer in L41 mutants than wild type. Lack of L41 caused slight hyperaccuracy (Table II) accompanied by an increase in the resistance to paromomycin. Also, whereas the ratio 60:40 S remained stable, there was a decrease in the amount of 80 S ribosomes. Because this was not accompanied by a similar decrease in the amount of polysomes, it could imply that L41 is not involved in the initiation phase of protein synthesis, a notion that needs further evaluation.The most pronounced effects of L41 are on the ribosomal peptidyltransferase activity (Table III and Fig. 4) and the translocation process of protein synthesis (Figs. 6 and 7). The peptidyltransferase activity of the ribosome was lowered 3-fold in the absence of L41. Likewise, the catalytic activity of the quintuple mutant was substantially lowered relative to that of the triple mutant. These results indicate a significant role for L41 in peptide bond formation. The presence of this dispensable protein is required so that the ribosome can exhibit its full catalytic activity. Because the ribosome is a ribozyme, it can only be surmised that L41 exerts its effect indirectly, possibly via allosteric interactions.The absence of L41 allowed increased elongation factor-independent (spontaneous) translocation. Also, the absence of L41 apparently increased the efficiency of elongation factor-dependent translocation, as shown by the lower amounts of EF2 needed to achieve 50% translocation. These data indicate that L41 may belong to a class of ribosomal proteins with a distinct role, that of preventing translocation from occurring spontaneously.Consistent with the role of L41 in translocation is the fact that its absence increased resistance to cycloheximide, a translocation inhibitor (Figs. 5 and 7). These results also suggest that yeast L41 participates in the binding site of cycloheximide on the ribosome. Mutations in two other ribosomal proteins, L42 (39Stevens D.R. Atteia A. Franzèn L.-G. Purton S. Mol. Gen. Genet. 2001; 264: 790-795Google Scholar) and L28 (40Kawai S. Mirato S. Mochizuki M. Shibuya I. Yano K. Takagi M. J. Bacteriol. 1992; 174: 254-262Google Scholar), were also shown to affect cycloheximide resistance. Subsequently, it was suggested that these two proteins may also play a central role in forming the cycloheximide binding site on the 60 S subunit (39Stevens D.R. Atteia A. Franzèn L.-G. Purton S. Mol. Gen. Genet. 2001; 264: 790-795Google Scholar).Multiple deletion mutants are useful in the study of ribosomal assembly and function. A quintuple mutant carrying a deletion of the two genes encoding L41, the two genes encoding L24, and the single gene encoding L39 permitted the cells to remain viable and functioning. The fact that each one of the three proteins is not essential does not necessarily mean that the phenotype obtained from the five gene deletions should have been expected; the three deficiencies together might have resulted in a lethal phenotype. This mutant provided also an alternative way to study the functions of protein L41. This was achieved by comparing the changes that ribosomes from these quintuple mutant cells undergo to those from a triple mutant lacking only L24 and L39. The comparative study of these two mutants reaffirmed the earlier findings that L41 has a limited impact on cell growth, association of ribosomal subunits, translational accuracy, and resistance to paromomycin, but does not affect cell viability or polyphenylalanine synthesis.A-site binding was significantly increased in the quintuple mutant compared with wild type (Table III). It has been suggested that an increase of A-site binding may arise from a higher affinity for accepting noncognate tRNAs and leads to a higher level of translational errors (41Karimi R. Ehrenberg M. EMBO J. 1996; 15: 1149-1154Google Scholar, 42Lodmell J.S. Dahlberg A.E. Science. 1997; 277: 1262-1267Google Scholar). Because the quintuple mutant exhibited a high translational error rate (Table II), our results are in agreement with this suggestion. In contrast, lack of L41 affected A-site binding slightly. Thus, the quintuple mutant strain exhibited slightly lower A-site binding over the triple mutant and so did the L41 mutant over the wild type, in agreement with the fact that L41 causes slight hyperaccuracy.It is interesting to note that the absence of L24 and L39 did not render the ribosome more susceptible to the absence of L41. In fact, the differences observed between L41 mutant and wild type were very similar to those between quintuple and triple mutants and fully accounted for by the absence of L41 alone in each of the two cases.The quintuple mutant YKS121 provides not only a useful tool with which to investigate the role of ribosomal protein L41; it also provides a measure of the degree of deterioration of the vital activities a eukaryotic cell can withstand. It is shown that cells tolerate at least a 4-fold decrease in the rate of protein synthesis as measured by the puromycin reaction (Table III) combined with a 3½-fold decrease in the fidelity of translation (Table II).It is worth mentioning that polyphenylalanine synthesis is marginally affected in the L41 mutants over the wild type or in the quintuple over the triple mutants, whereas peptidyltransferase activity is lowered 3- and 2-fold, respectively, although peptide bond formation is a reaction of the elongation cycle of protein synthesis. A similar effect,i.e. a significant reduction of peptide bond formation but much less impairment of polyphenylalanine synthesis has been observed with a series of 23 S rRNA mutations (43Spahn C.M. Schafer M.A. Krayevsky A.A. Nierhaus K.H. J. Biol. Chem. 1996; 271: 32857-32862Google Scholar) or with ribosomal protein L2 (23Diedrich G. Spahn C.M.T. Stelzl U. Schäfer M.A. Wooten T. Bochkariov D.E. Cooperman B.S. Traut R.R. Nierhaus K.H. EMBO J. 2000; 19: 5241-5250Google Scholar). These results may be explained by the hypothesis that the two processes have different rate-limiting steps. In fact the rate-limiting step of the elongation cycle is the occupation of the A-site and this is much slower than peptidyl transfer (44Bilgin N. Ehrenberg M. Kurland C. FEBS Lett. 1988; 233: 95-99Google Scholar, 45Schilling-Bartetzko S. Bartetzko A. Nierhaus K.H. J. Biol. Chem. 1992; 267: 4703-4712Google Scholar). For the puromycin reaction, however, the rate-limiting step is the peptide bond formation and not the binding of puromycin to the A-site (23Diedrich G. Spahn C.M.T. Stelzl U. Schäfer M.A. Wooten T. Bochkariov D.E. Cooperman B.S. Traut R.R. Nierhaus K.H. EMBO J. 2000; 19: 5241-5250Google Scholar). As a result, a significant reduction in the rate of peptidyltransferase activity would more strongly decrease the rate of the puromycin reaction than the rate of polyphenylalanine synthesis.In conclusion, the absence of protein L41 affected in varying degrees three of the main activities of the ribosome, i.e.peptide bond formation, translocation, and decoding, adding to the notion that these activities are interrelated and that some ribosomal proteins may possess more than one ribosomal function. Similar observations have been made recently for other ribosomal proteins, such as L39, the absence of which decreased translational fidelity but increased somewhat the ribosomal peptidyltransferase activity (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar, 24Dresios J. Panopoulos P. Frantziou C.P. Synetos D. Biochemistry. 2001; 40: 8101-8108Google Scholar). Likewise, several deleterious mutations in domain V of 23 S rRNA (9O'Connor M. Lee W-C.M. Mankad A. Squires C.L. Dahlberg A.E. Nucleic Acids Res. 2001; 29: 710-715Google Scholar) have been linked with peptide bond formation whereas they also affect the fidelity of decoding, an activity of the small ribosomal subunit. Moreover, a model has been proposed in Escherichia coliwhereby elongation factor G promotes translocation by modulating the communication between the peptidyltransferase domain of 23 S rRNA and the decoding region of 16 S rRNA during elongation (46Koosha H. Cameron D. Andrews K. Dalberg A.E. March P.E. RNA (N. Y.). 2000; 6: 1166-1173Google Scholar). This communication may be achieved in various ways; for example, signals from the decoding center on the small subunit to the peptidyltransferase center on the large subunit can be transmitted either by ligands that contact both regions, e.g. bound tRNAs, or by the intersubunit bridges that connect the subunits (47Spahn C.M.T. Beckmann R. Eswar N. Penczek P.A. Sali A. Blobel G. Frank J. Cell. 2001; 107: 373-386Google Scholar). The ribosome is a large macromolecular machine that consists of a large number of proteins and several molecules of ribosomal RNA (rRNA). The ribosome is responsible for the translation of the genetic message, which results in protein synthesis. The crystal structures of the 30 S (1Wimberly B.T. Brodersen D.E. Clemons W.M. Morgan-Warren R.J. Carter A.P. Vonrhein C. Hartsch T. Ramakrishnan V. Nature. 2000; 407: 327-348Google Scholar, 2Schluenzen F. Tocilj A. Zarivach R. Harms J. Gluehmann M. Janell D. Bashan A. Bartels H. Agmon I. Franceschi F. Yonath A. Cell. 2000; 102: 615-623Google Scholar) and 50 S (3Ban N. Nissen P. Hansen J. Moore P.B. Steitz T.A. Science. 2000; 289: 905-920Google Scholar, 4Harms J. Schluenzen F. Zarivach R. Bashan A. Gat S. Agmon I. Bartels H. Franceschi F. Yonath A. Cell. 2001; 107: 679-688Google Scholar) ribosomal subunits, and the intact 70 S ribosome (5Yusupov M.M. Yusupova G.Z. Baucom A. Lieberman K. Earnest T.N. Cate J.H. Noller H.F. Science. 2001; 292: 883-896Google Scholar) are contributing to a better understanding of ribosomal function. It has now been confirmed that rRNA plays the major role in ribosomal structure and function, including the two most important activities of the ribosome: the decoding process and the peptidyltransferase activity. The 16 S rRNA of the small ribosomal subunit is responsible for the decoding process, the selection of the cognate tRNA (6Carter A.P. Clemons W.M., Jr. Brodersen D.E. Morgan-Warren R.J. Wimberly B.T. Ramakrishnan V. Nature. 2000; 407: 340-348Google Scholar). The central loop of domain V of 23 S rRNA of the large ribosomal subunit is responsible for the catalytic activity of the ribosome (7Nissen P. Hansen J. Ban N. Moore P.B. Steitz T.A. Science. 2000; 289: 920-930Google Scholar, 8Muth G.W. Ortoleva–Donnelly L. Strobel S.A. Science. 2000; 289: 947-950Google Scholar). The search is still underway to identify a critical site or nucleotide(s) of 23 S rRNA involved in the catalysis of peptide bond formation (9O'Connor M. Lee W-C.M. Mankad A. Squires C.L. Dahlberg A.E. Nucleic Acids Res. 2001; 29: 710-715Google Scholar, 10Bayfield M.A. Dahlberg E.A. Schulmeister U. Dorner S. Barta A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10096-10101Google Scholar, 11Xiong L. Polacek N. Sander P. Böttger E.C. Mankin A. RNA. 2001; 7: 1365-1369Google Scholar, 12Thompson J. Kim D.F. O'Connor M. Lieberman K.R. Bayfield M.A. Gregory S.T. Green R. Noller H.F. Dahlberg A.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9002-9007Google Scholar, 13Schmeing T.M. Seila A.C. Hansen J.L. Freeborn B. Soukup J.K. Scaringe S.A. Strobel S.A. Moore P.B. Steitz T.A. Nat. Struct. Biol. 2002; 9: 225-230Google Scholar). It is still possible that the rate of peptide bond formation is influenced by structural rearrangements of RNA in the peptidyltransferase center that would affect the positioning of the substrates (14Polacek N. Gaynor M. Yassin A. Mankin A.S. Nature. 2001; 411: 498-501Google Scholar). Ribosomal proteins offer structural support to the ribosome by stabilizing and orienting the ribosomal RNA into a specific, active structure (15Cech T.R. Science. 2000; 289: 878-879Google Scholar). Also, they are crucial for the assembly of functional ribosomes (16Stern S. Powers T. Changchien L.M. Noller H.F. Science. 1989; 244: 783-790Google Scholar). Several ribosomal proteins of the small subunit (17Vincent A. Liebman S.W. Genetics. 1992; 132: 375-386Google Scholar, 18Alksne L.E. Anthony R.A. Liebman S.W. Warner J.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9538-9541Google Scholar, 19Liebman S.W. Chernoff Y.O. Liu R. Biochem. Cell Biol. 1995; 73: 1141-1149Google Scholar, 20Synetos D. Frantziou C.P. Alksne L.E. Biochim. Biophys. Acta. 1996; 1309: 156-166Google Scholar) and at least one of the large subunit (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar) appear to affect the decoding process. Moreover, some ribosomal proteins appear to influence the peptidyltransferase activity of the ribosome (22Wower I.K. Wower J. Zimmermann R.A. J. Biol. Chem. 1998; 273: 19847-19852Google Scholar, 23Diedrich G. Spahn C.M.T. Stelzl U. Schäfer M.A. Wooten T. Bochkariov D.E. Cooperman B.S. Traut R.R. Nierhaus K.H. EMBO J. 2000; 19: 5241-5250Google Scholar, 24Dresios J. Panopoulos P. Frantziou C.P. Synetos D. Biochemistry. 2001; 40: 8101-8108Google Scholar). Yeast ribosomal protein L41 is the smallest and the most basic eukaryotic protein. Its open reading frame is composed of only 25 amino acids, 17 of which are arginines or lysines (25Suzuki K. Hashimoto T. Otaka E. Curr. Genet. 1990; 17: 185-190Google Scholar). L41 is highly conserved in eukaryotes and it is present in certain archaea,e.g. Methanococcus jannaschii, but not in eubacteria. A protein with similar properties is present in certain thermophilic eubacteria, e.g. Thermus thermophilus (26Choli T. Franceschi F. Wittmann-Liebold B. Yonath A. Biol. Chem. Hoppe-Seyler. 1993; 374: 377-383Google Scholar). In Saccharomyces cerevisiae, L41 is encoded by two genes, RPL41A and RPL41B (25Suzuki K. Hashimoto T. Otaka E. Curr. Genet. 1990; 17: 185-190Google Scholar). In a recent study, the properties and the translation of the mRNAs encoding L41 were investigated (27Yu X. Warner J.R. J. Biol. Chem. 2001; 276: 33821-33825Google Scholar). It was found that the L41 mRNAs are translated exclusively on monosomes and the entire translation process from initiation through to termination occurs in about 2 s. However, as with most ribosomal proteins, the function of L41 remains unknown (27Yu X. Warner J.R. J. Biol. Chem. 2001; 276: 33821-33825Google Scholar). There is evidence that suggests that protein L41 may possess extraribosomal activity; it associates directly in vitrowith subunit b of protein kinase CKII, although it is not a substrate for CKII phosphorylation. However, L41 protein stimulates the phosphorylation of DNA topoisomerase IIa by CKII. Additionally, L41 enhances the autophosphorylation of CKIIa (28Lee J.H. Kim J.M. Lee Y.T. Marshak D.R. Bae Y.S. Biochem. Biophys. Res. Commun. 1997; 238: 462-467Google Scholar). These data indicate that L41 associates with CKII and can modulate its activity toward a specific substrate(s). In the present work we tried to gain insight into the role of protein L41 in protein synthesis. For this purpose we examined two yeast strains. The first lacked the two genes encoding L41. The second strain lacked L41 in addition to the absence of L24 and L39, two eukaryote-specific proteins studied recently (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar, 24Dresios J. Panopoulos P. Frantziou C.P. Synetos D. Biochemistry. 2001; 40: 8101-8108Google Scholar). The fact that the cells remained viable upon removal of the two genes encoding L41 in addition to the absence of the two genes encoding L24 and the single gene encoding L39, allowed for the first time the study of a quintuple mutant in yeast as well as the study of the role of L41 in combination with the effects of the other two proteins. Our results suggest that a dispensable ribosomal protein such as L41 may affect in varying degrees several important functions of yeast ribosomes confirming the interconnectedness of these ribosomal activities. DISCUSSIONIn an effort to contribute to the understanding of yeast ribosomal structure and function, we examined the role(s) of ribosomal protein L41. For this purpose we used two strains, one in which the two genes for L41 were deleted and a second strain, in which the two genes for L41 were deleted from a background already lacking the genes for L24 and L39. We have found that in the absence of L24 and L39 the cell exhibited reduced protein synthesis activity and decreased translational accuracy, two closely related functions (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar). Thus, this was an appropriate background in which to study the effect of the absence of a third protein, L41, on the above functions.We found that protein L41 is dispensable for the yeast S. cerevisiae. This is the third case of a eukaryotic ribosomal protein, not found in eubacteria, which is nonessential for cell viability. The other two nonessential proteins, L24 and L39, were found to have significant effects on certain parameters of protein synthesis (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar, 24Dresios J. Panopoulos P. Frantziou C.P. Synetos D. Biochemistry. 2001; 40: 8101-8108Google Scholar). Specifically, L24 acted on the assembly of ribosomes and the kinetics of protein synthesis, whereas L39 acted on the accuracy of translation and the assembly of the 60 S subunits. Protein L41 is dispensable but it is not entirely innocuous to the cell: doubling times were somewhat longer in L41 mutants than wild type. Lack of L41 caused slight hyperaccuracy (Table II) accompanied by an increase in the resistance to paromomycin. Also, whereas the ratio 60:40 S remained stable, there was a decrease in the amount of 80 S ribosomes. Because this was not accompanied by a similar decrease in the amount of polysomes, it could imply that L41 is not involved in the initiation phase of protein synthesis, a notion that needs further evaluation.The most pronounced effects of L41 are on the ribosomal peptidyltransferase activity (Table III and Fig. 4) and the translocation process of protein synthesis (Figs. 6 and 7). The peptidyltransferase activity of the ribosome was lowered 3-fold in the absence of L41. Likewise, the catalytic activity of the quintuple mutant was substantially lowered relative to that of the triple mutant. These results indicate a significant role for L41 in peptide bond formation. The presence of this dispensable protein is required so that the ribosome can exhibit its full catalytic activity. Because the ribosome is a ribozyme, it can only be surmised that L41 exerts its effect indirectly, possibly via allosteric interactions.The absence of L41 allowed increased elongation factor-independent (spontaneous) translocation. Also, the absence of L41 apparently increased the efficiency of elongation factor-dependent translocation, as shown by the lower amounts of EF2 needed to achieve 50% translocation. These data indicate that L41 may belong to a class of ribosomal proteins with a distinct role, that of preventing translocation from occurring spontaneously.Consistent with the role of L41 in translocation is the fact that its absence increased resistance to cycloheximide, a translocation inhibitor (Figs. 5 and 7). These results also suggest that yeast L41 participates in the binding site of cycloheximide on the ribosome. Mutations in two other ribosomal proteins, L42 (39Stevens D.R. Atteia A. Franzèn L.-G. Purton S. Mol. Gen. Genet. 2001; 264: 790-795Google Scholar) and L28 (40Kawai S. Mirato S. Mochizuki M. Shibuya I. Yano K. Takagi M. J. Bacteriol. 1992; 174: 254-262Google Scholar), were also shown to affect cycloheximide resistance. Subsequently, it was suggested that these two proteins may also play a central role in forming the cycloheximide binding site on the 60 S subunit (39Stevens D.R. Atteia A. Franzèn L.-G. Purton S. Mol. Gen. Genet. 2001; 264: 790-795Google Scholar).Multiple deletion mutants are useful in the study of ribosomal assembly and function. A quintuple mutant carrying a deletion of the two genes encoding L41, the two genes encoding L24, and the single gene encoding L39 permitted the cells to remain viable and functioning. The fact that each one of the three proteins is not essential does not necessarily mean that the phenotype obtained from the five gene deletions should have been expected; the three deficiencies together might have resulted in a lethal phenotype. This mutant provided also an alternative way to study the functions of protein L41. This was achieved by comparing the changes that ribosomes from these quintuple mutant cells undergo to those from a triple mutant lacking only L24 and L39. The comparative study of these two mutants reaffirmed the earlier findings that L41 has a limited impact on cell growth, association of ribosomal subunits, translational accuracy, and resistance to paromomycin, but does not affect cell viability or polyphenylalanine synthesis.A-site binding was significantly increased in the quintuple mutant compared with wild type (Table III). It has been suggested that an increase of A-site binding may arise from a higher affinity for accepting noncognate tRNAs and leads to a higher level of translational errors (41Karimi R. Ehrenberg M. EMBO J. 1996; 15: 1149-1154Google Scholar, 42Lodmell J.S. Dahlberg A.E. Science. 1997; 277: 1262-1267Google Scholar). Because the quintuple mutant exhibited a high translational error rate (Table II), our results are in agreement with this suggestion. In contrast, lack of L41 affected A-site binding slightly. Thus, the quintuple mutant strain exhibited slightly lower A-site binding over the triple mutant and so did the L41 mutant over the wild type, in agreement with the fact that L41 causes slight hyperaccuracy.It is interesting to note that the absence of L24 and L39 did not render the ribosome more susceptible to the absence of L41. In fact, the differences observed between L41 mutant and wild type were very similar to those between quintuple and triple mutants and fully accounted for by the absence of L41 alone in each of the two cases.The quintuple mutant YKS121 provides not only a useful tool with which to investigate the role of ribosomal protein L41; it also provides a measure of the degree of deterioration of the vital activities a eukaryotic cell can withstand. It is shown that cells tolerate at least a 4-fold decrease in the rate of protein synthesis as measured by the puromycin reaction (Table III) combined with a 3½-fold decrease in the fidelity of translation (Table II).It is worth mentioning that polyphenylalanine synthesis is marginally affected in the L41 mutants over the wild type or in the quintuple over the triple mutants, whereas peptidyltransferase activity is lowered 3- and 2-fold, respectively, although peptide bond formation is a reaction of the elongation cycle of protein synthesis. A similar effect,i.e. a significant reduction of peptide bond formation but much less impairment of polyphenylalanine synthesis has been observed with a series of 23 S rRNA mutations (43Spahn C.M. Schafer M.A. Krayevsky A.A. Nierhaus K.H. J. Biol. Chem. 1996; 271: 32857-32862Google Scholar) or with ribosomal protein L2 (23Diedrich G. Spahn C.M.T. Stelzl U. Schäfer M.A. Wooten T. Bochkariov D.E. Cooperman B.S. Traut R.R. Nierhaus K.H. EMBO J. 2000; 19: 5241-5250Google Scholar). These results may be explained by the hypothesis that the two processes have different rate-limiting steps. In fact the rate-limiting step of the elongation cycle is the occupation of the A-site and this is much slower than peptidyl transfer (44Bilgin N. Ehrenberg M. Kurland C. FEBS Lett. 1988; 233: 95-99Google Scholar, 45Schilling-Bartetzko S. Bartetzko A. Nierhaus K.H. J. Biol. Chem. 1992; 267: 4703-4712Google Scholar). For the puromycin reaction, however, the rate-limiting step is the peptide bond formation and not the binding of puromycin to the A-site (23Diedrich G. Spahn C.M.T. Stelzl U. Schäfer M.A. Wooten T. Bochkariov D.E. Cooperman B.S. Traut R.R. Nierhaus K.H. EMBO J. 2000; 19: 5241-5250Google Scholar). As a result, a significant reduction in the rate of peptidyltransferase activity would more strongly decrease the rate of the puromycin reaction than the rate of polyphenylalanine synthesis.In conclusion, the absence of protein L41 affected in varying degrees three of the main activities of the ribosome, i.e.peptide bond formation, translocation, and decoding, adding to the notion that these activities are interrelated and that some ribosomal proteins may possess more than one ribosomal function. Similar observations have been made recently for other ribosomal proteins, such as L39, the absence of which decreased translational fidelity but increased somewhat the ribosomal peptidyltransferase activity (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar, 24Dresios J. Panopoulos P. Frantziou C.P. Synetos D. Biochemistry. 2001; 40: 8101-8108Google Scholar). Likewise, several deleterious mutations in domain V of 23 S rRNA (9O'Connor M. Lee W-C.M. Mankad A. Squires C.L. Dahlberg A.E. Nucleic Acids Res. 2001; 29: 710-715Google Scholar) have been linked with peptide bond formation whereas they also affect the fidelity of decoding, an activity of the small ribosomal subunit. Moreover, a model has been proposed in Escherichia coliwhereby elongation factor G promotes translocation by modulating the communication between the peptidyltransferase domain of 23 S rRNA and the decoding region of 16 S rRNA during elongation (46Koosha H. Cameron D. Andrews K. Dalberg A.E. March P.E. RNA (N. Y.). 2000; 6: 1166-1173Google Scholar). This communication may be achieved in various ways; for example, signals from the decoding center on the small subunit to the peptidyltransferase center on the large subunit can be transmitted either by ligands that contact both regions, e.g. bound tRNAs, or by the intersubunit bridges that connect the subunits (47Spahn C.M.T. Beckmann R. Eswar N. Penczek P.A. Sali A. Blobel G. Frank J. Cell. 2001; 107: 373-386Google Scholar). In an effort to contribute to the understanding of yeast ribosomal structure and function, we examined the role(s) of ribosomal protein L41. For this purpose we used two strains, one in which the two genes for L41 were deleted and a second strain, in which the two genes for L41 were deleted from a background already lacking the genes for L24 and L39. We have found that in the absence of L24 and L39 the cell exhibited reduced protein synthesis activity and decreased translational accuracy, two closely related functions (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar). Thus, this was an appropriate background in which to study the effect of the absence of a third protein, L41, on the above functions. We found that protein L41 is dispensable for the yeast S. cerevisiae. This is the third case of a eukaryotic ribosomal protein, not found in eubacteria, which is nonessential for cell viability. The other two nonessential proteins, L24 and L39, were found to have significant effects on certain parameters of protein synthesis (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar, 24Dresios J. Panopoulos P. Frantziou C.P. Synetos D. Biochemistry. 2001; 40: 8101-8108Google Scholar). Specifically, L24 acted on the assembly of ribosomes and the kinetics of protein synthesis, whereas L39 acted on the accuracy of translation and the assembly of the 60 S subunits. Protein L41 is dispensable but it is not entirely innocuous to the cell: doubling times were somewhat longer in L41 mutants than wild type. Lack of L41 caused slight hyperaccuracy (Table II) accompanied by an increase in the resistance to paromomycin. Also, whereas the ratio 60:40 S remained stable, there was a decrease in the amount of 80 S ribosomes. Because this was not accompanied by a similar decrease in the amount of polysomes, it could imply that L41 is not involved in the initiation phase of protein synthesis, a notion that needs further evaluation. The most pronounced effects of L41 are on the ribosomal peptidyltransferase activity (Table III and Fig. 4) and the translocation process of protein synthesis (Figs. 6 and 7). The peptidyltransferase activity of the ribosome was lowered 3-fold in the absence of L41. Likewise, the catalytic activity of the quintuple mutant was substantially lowered relative to that of the triple mutant. These results indicate a significant role for L41 in peptide bond formation. The presence of this dispensable protein is required so that the ribosome can exhibit its full catalytic activity. Because the ribosome is a ribozyme, it can only be surmised that L41 exerts its effect indirectly, possibly via allosteric interactions. The absence of L41 allowed increased elongation factor-independent (spontaneous) translocation. Also, the absence of L41 apparently increased the efficiency of elongation factor-dependent translocation, as shown by the lower amounts of EF2 needed to achieve 50% translocation. These data indicate that L41 may belong to a class of ribosomal proteins with a distinct role, that of preventing translocation from occurring spontaneously. Consistent with the role of L41 in translocation is the fact that its absence increased resistance to cycloheximide, a translocation inhibitor (Figs. 5 and 7). These results also suggest that yeast L41 participates in the binding site of cycloheximide on the ribosome. Mutations in two other ribosomal proteins, L42 (39Stevens D.R. Atteia A. Franzèn L.-G. Purton S. Mol. Gen. Genet. 2001; 264: 790-795Google Scholar) and L28 (40Kawai S. Mirato S. Mochizuki M. Shibuya I. Yano K. Takagi M. J. Bacteriol. 1992; 174: 254-262Google Scholar), were also shown to affect cycloheximide resistance. Subsequently, it was suggested that these two proteins may also play a central role in forming the cycloheximide binding site on the 60 S subunit (39Stevens D.R. Atteia A. Franzèn L.-G. Purton S. Mol. Gen. Genet. 2001; 264: 790-795Google Scholar). Multiple deletion mutants are useful in the study of ribosomal assembly and function. A quintuple mutant carrying a deletion of the two genes encoding L41, the two genes encoding L24, and the single gene encoding L39 permitted the cells to remain viable and functioning. The fact that each one of the three proteins is not essential does not necessarily mean that the phenotype obtained from the five gene deletions should have been expected; the three deficiencies together might have resulted in a lethal phenotype. This mutant provided also an alternative way to study the functions of protein L41. This was achieved by comparing the changes that ribosomes from these quintuple mutant cells undergo to those from a triple mutant lacking only L24 and L39. The comparative study of these two mutants reaffirmed the earlier findings that L41 has a limited impact on cell growth, association of ribosomal subunits, translational accuracy, and resistance to paromomycin, but does not affect cell viability or polyphenylalanine synthesis. A-site binding was significantly increased in the quintuple mutant compared with wild type (Table III). It has been suggested that an increase of A-site binding may arise from a higher affinity for accepting noncognate tRNAs and leads to a higher level of translational errors (41Karimi R. Ehrenberg M. EMBO J. 1996; 15: 1149-1154Google Scholar, 42Lodmell J.S. Dahlberg A.E. Science. 1997; 277: 1262-1267Google Scholar). Because the quintuple mutant exhibited a high translational error rate (Table II), our results are in agreement with this suggestion. In contrast, lack of L41 affected A-site binding slightly. Thus, the quintuple mutant strain exhibited slightly lower A-site binding over the triple mutant and so did the L41 mutant over the wild type, in agreement with the fact that L41 causes slight hyperaccuracy. It is interesting to note that the absence of L24 and L39 did not render the ribosome more susceptible to the absence of L41. In fact, the differences observed between L41 mutant and wild type were very similar to those between quintuple and triple mutants and fully accounted for by the absence of L41 alone in each of the two cases. The quintuple mutant YKS121 provides not only a useful tool with which to investigate the role of ribosomal protein L41; it also provides a measure of the degree of deterioration of the vital activities a eukaryotic cell can withstand. It is shown that cells tolerate at least a 4-fold decrease in the rate of protein synthesis as measured by the puromycin reaction (Table III) combined with a 3½-fold decrease in the fidelity of translation (Table II). It is worth mentioning that polyphenylalanine synthesis is marginally affected in the L41 mutants over the wild type or in the quintuple over the triple mutants, whereas peptidyltransferase activity is lowered 3- and 2-fold, respectively, although peptide bond formation is a reaction of the elongation cycle of protein synthesis. A similar effect,i.e. a significant reduction of peptide bond formation but much less impairment of polyphenylalanine synthesis has been observed with a series of 23 S rRNA mutations (43Spahn C.M. Schafer M.A. Krayevsky A.A. Nierhaus K.H. J. Biol. Chem. 1996; 271: 32857-32862Google Scholar) or with ribosomal protein L2 (23Diedrich G. Spahn C.M.T. Stelzl U. Schäfer M.A. Wooten T. Bochkariov D.E. Cooperman B.S. Traut R.R. Nierhaus K.H. EMBO J. 2000; 19: 5241-5250Google Scholar). These results may be explained by the hypothesis that the two processes have different rate-limiting steps. In fact the rate-limiting step of the elongation cycle is the occupation of the A-site and this is much slower than peptidyl transfer (44Bilgin N. Ehrenberg M. Kurland C. FEBS Lett. 1988; 233: 95-99Google Scholar, 45Schilling-Bartetzko S. Bartetzko A. Nierhaus K.H. J. Biol. Chem. 1992; 267: 4703-4712Google Scholar). For the puromycin reaction, however, the rate-limiting step is the peptide bond formation and not the binding of puromycin to the A-site (23Diedrich G. Spahn C.M.T. Stelzl U. Schäfer M.A. Wooten T. Bochkariov D.E. Cooperman B.S. Traut R.R. Nierhaus K.H. EMBO J. 2000; 19: 5241-5250Google Scholar). As a result, a significant reduction in the rate of peptidyltransferase activity would more strongly decrease the rate of the puromycin reaction than the rate of polyphenylalanine synthesis. In conclusion, the absence of protein L41 affected in varying degrees three of the main activities of the ribosome, i.e.peptide bond formation, translocation, and decoding, adding to the notion that these activities are interrelated and that some ribosomal proteins may possess more than one ribosomal function. Similar observations have been made recently for other ribosomal proteins, such as L39, the absence of which decreased translational fidelity but increased somewhat the ribosomal peptidyltransferase activity (21Dresios J. Derkatch I.L. Liebman S.W. Synetos D. Biochemistry. 2000; 39: 7236-7244Google Scholar, 24Dresios J. Panopoulos P. Frantziou C.P. Synetos D. Biochemistry. 2001; 40: 8101-8108Google Scholar). Likewise, several deleterious mutations in domain V of 23 S rRNA (9O'Connor M. Lee W-C.M. Mankad A. Squires C.L. Dahlberg A.E. Nucleic Acids Res. 2001; 29: 710-715Google Scholar) have been linked with peptide bond formation whereas they also affect the fidelity of decoding, an activity of the small ribosomal subunit. Moreover, a model has been proposed in Escherichia coliwhereby elongation factor G promotes translocation by modulating the communication between the peptidyltransferase domain of 23 S rRNA and the decoding region of 16 S rRNA during elongation (46Koosha H. Cameron D. Andrews K. Dalberg A.E. March P.E. RNA (N. Y.). 2000; 6: 1166-1173Google Scholar). This communication may be achieved in various ways; for example, signals from the decoding center on the small subunit to the peptidyltransferase center on the large subunit can be transmitted either by ligands that contact both regions, e.g. bound tRNAs, or by the intersubunit bridges that connect the subunits (47Spahn C.M.T. Beckmann R. Eswar N. Penczek P.A. Sali A. Blobel G. Frank J. Cell. 2001; 107: 373-386Google Scholar). We thank Professor Jon Warner for making several yeast strains available to us. We also thank Dr. Irina L. Derkatch for providing strain 38C. We are indebted to Dr. Albert Sitikov for the generous gift of EF2. We also thank Dr. Theodora Choli-Papadopoulou for active interest and help and Dr. Dimitrios Kalpaxis for many helpful discussions and critical reading of the manuscript.
Hydrogen sulfide (H 2 S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H 2 S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H 2 S and NO exert their effects via separate pathways, the results of the current study show that H 2 S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H 2 S increases intracellular cyclic guanosine 5′-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein ( VASP ). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H 2 S-stimulated angiogenic response, and attenuated H 2 S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H 2 S signaling. Conversely, silencing of the H 2 S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H 2 S in the vascular activity of NO. The actions of H 2 S and NO converge at cGMP; though H 2 S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H 2 S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H 2 S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H 2 S are mutually required for the physiological control of vascular function.
We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5' leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5' leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.
The effect of somatostatin on nerve‐induced vasodilatation and the release of substance P (SP) was studied in the dental pulp of anesthetized cats. Changes in pulpal blood flow were determined by measuring the rate of disappearance of a local depot of radioactive tracer. The release of SP was studied indirectly by determining the residual amounts of substance P‐like immunoreactivity (SPLI)in the pulps by radioimmunoassay. Electrical stimulation (3 min at 10 V, 15 Hz and 5 ms) of the distal end of the cut inferior alveolar nerve (IAN) increased pulpal blood flow. After pretreatment (10 min) with somatostatin (30 pmol/min) similar nerve stimulation was without effect on pulpal blood flow. Intra‐arterial infusion of somatostatin (30 pmol/min) had no effect on pulpal blood flow and did not influence the vasodilator response to SP. Following IAN stimulation (3–45 min) and subsequent incubation (30 min, 37°C) of the lower canine teeth, the SPLI levels in ipsilateral pulps were significantly lower (47.5% reduction) than those in contralateral, unstimulated controls. In cats pretreated with somatostatin (30 pmol/min for 10 min, i.a.) similar nerve stimulation (3 min) did not reduce the pulpal SPLI levels as compared to controls. The results show that nerve‐induced vasodilatation and release of SPLI are inhibited by somatostatin. They are consistent with the hypothesis that vasodilatation in the cat dental pulp produced by stimulation of the IAN is mediated by substance P.
Ammonia and organic acids constitute a major part of the bacterial metabolites formed in carious decay. The aim of the present study was to investigate their effect on the intradental sensory nerves. Nerve impulse activity was recorded from canine teeth in cats after application of the test solutions in deep dentinal cavities. Ammonia (17—134 mM) consistently generated nerve impulses, whereas organic acids (0.001—1 M) failed to induce any impulse activity. In contrast, acid application resulted in an inhibition of the ongoing nerve activity induced by various stimuli (hypertonic NaCl solution, mechanical pulp exposure, and compound 48/80). However, acid treatment of the cavities resulted in an enhanced neural response to ammonia stimulation. Thus, the present results demonstrate that these bacterial metabolites can influence intradental sensory nerve activity. It is suggested that they may also modulate the symptoms from decayed teeth.
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