Fluorophores at the N Terminus of Nascent Chloramphenicol Acetyltransferase Peptides Affect Translation and Movement through the Ribosome

Abstract

Structurally different fluorescent probes were covalently attached to methionyl-tRNAf and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [35S]Met-tRNAf. All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 × 11 Å, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNAf compared with fMet-tRNAf with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA. Structurally different fluorescent probes were covalently attached to methionyl-tRNAf and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [35S]Met-tRNAf. All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 × 11 Å, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNAf compared with fMet-tRNAf with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA. E. coli initiator tRNA aminoacylated with methionine, the amino group of which is formylated chloramphenicol acetyltransferase cytidylic acid-puromycin 3,3-dithiobis(sulfosuccinimidyl)propionate high performance liquid chromatography polyacrylamide gel electrophoresis rhodanese N-tris(hydroxymethyl)methylglycine Initiation of protein synthesis by a specific initiator tRNAMet at an AUG codon is a universally conserved step in gene expression for both eukaryotes and prokaryotes. In prokaryotes, methionine linked to the initiator tRNAMet is formylated to give fMet-tRNAf.1However, peptide initiation can be carried out with otherN-acyl derivatives of Met-tRNAf, many of which can be synthesized chemically from Met-tRNAf. We have synthesized coumarin maleimide-S-acetyl-Met-tRNAf to incorporate a fluorescent probe at the N terminus of polypeptides during their synthesis in a cell-free coupled transcription/translation system derived from Escherichia coli (1.Kudlicki W. Odom O.W. Kramer G. Hardesty B. J. Mol. Biol. 1994; 244: 319-331Crossref PubMed Scopus (52) Google Scholar, 2.Hardesty B. Kudlicki W. Odom O.W. Zhang T. McCarthy D. Kramer G. Biochem. Cell Biol. 1995; 73: 1199-1207Crossref PubMed Scopus (16) Google Scholar, 3.Kudlicki W. Kitaoka Y. Odom O.W. Kramer G. Hardesty B. J. Mol. Biol. 1995; 252: 203-212Crossref PubMed Scopus (24) Google Scholar, 4.Tsalkova T. Odom O.W. Kramer G. Hardesty B. J. Mol. Biol. 1998; 278: 713-723Crossref PubMed Scopus (35) Google Scholar). A considerable amount of information on the role of the molecular chaperones DnaJ and DnaK in folding of the nascent protein and on cotranslational folding of nascent peptides was obtained through this method. In addition, we showed recently (5.Tsalkova T. Kramer G. Hardesty B. J. Mol. Biol. 1999; 286: 71-81Crossref PubMed Scopus (15) Google Scholar) that incorporation of coumarin at the N terminus of bacterial chloramphenicol acetyltransferase (CAT) resulted in increased ribosomal pausing at the already existing pause sites provided translation was slowed. Translational pause sites and their potential causes have been extensively discussed in this publication. The studies cited above have prompted the question of what are the limits in terms of size and chemical character of the modification on the N terminus of nascent peptides under optimal translation conditions. Will the ribosomes initiate with bulky (pyrene, eosin) or a charged group (cascade yellow) covalently attached to the α-amino group of methionine on the initiator-tRNA as efficiently as with fMet-tRNAf? Initiation of protein synthesis is usually the rate-controlling step and a major point for regulation of translation (6.Gualerzi C. Pon C. Biochemistry. 1990; 29: 5881-5889Crossref PubMed Scopus (402) Google Scholar). Do the fluorophores at the N terminus affect the amount and the rate at which polypeptides are formed after they are initiated? Are they incorporated into native full-length protein? The last two questions imply that the bulky modification at the N terminus may hinder the required folding of the nascent peptide to first pass through the tunnel inside the large ribosomal subunit (7.Frank J. J. Struct. Biol. 1998; 124: 142-150Crossref PubMed Scopus (19) Google Scholar), then to acquire the three-dimensional structure of the native protein. Here we report the effect on translation of three additional N-terminal fluorophores (pyrene, cascade yellow, eosin), which vary in size and structure compared with coumarin. Synthesis of bovine rhodanese (RHO, a thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) or bacterial CAT (EC2.3.1.28) was initiated in the presence of fluorophore-Met-tRNAf. Formation of nascent peptides and full-length product was analyzed and compared with polypeptides formed with N-formyl-methionine at the N terminus. The results indicate that RHO and CAT could be produced when pyrene-Met or cascade yellow-Met were at the N terminus of the protein; however, using eosin-Met-tRNAf, the translational machinery worked less efficiently. With all fluorophore-Met-tRNAf species, peptide initiation was an impaired step. With CAT, but not with RHO, pausing during translation was increased, resulting in accumulation of low molecular weight nascent peptides. The results are discussed under the aspect of folding of the nascent polypeptides on the ribosomes. 3-(4-Maleimidophenyl)-7-(diethylamino-4-methyl) coumarin,N-(1-pyrene)-maleimide, eosin-5-maleimide, and cascade yellow succinimidyl ester were from Molecular Probes, Inc. (Eugene, OR). The structures of coumarin-maleimide,N-(1-pyrene)-maleimide, and eosin-5-maleimide are given in Ref. 25.Haugland R. Handbook of Fluorescent Probes and Research Chemicals. 6th Ed. Molecular Probes, Inc., Eugene, OR1996Google Scholar. The structure of the succinimidyl ester of cascade yellow is given in Ref. 26.Molecular Probes Inc BioProbes 25. Molecular Probes, Inc., Eugene, OR1997Google Scholar. tRNAfMet, rifampicin, and all other biochemicals were from Sigma. [35S]Methionine was purchased from NEN Life Science Products. Puromycin-CPG and 5′-dimethoxytrityl-N-acetylcytidine, 2′-O-TBDMS-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite were purchased from Glen Research (Sterling, VA). Cytidylic acid-puromycin (C-puro) was synthesized from these components by Dr. John Lee (Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX). Low molecular weight protein standards for SDS-PAGE were from Promega (Madison, WI). The original rhodanese plasmid was a kind gift from Dr. Paul Horowitz (University of Texas Health Science Center, San Antonio, TX), and the plasmid containing CAT coding sequence was provided by Dr. A. S. Spirin (Institute of Protein Research, Pushchino, Russia). Both the coding sequences of rhodanese and CAT were inserted under the T7 promoter in a pGEM vector. Coumarin-SAc-Met-tRNAf was prepared as described previously (1.Kudlicki W. Odom O.W. Kramer G. Hardesty B. J. Mol. Biol. 1994; 244: 319-331Crossref PubMed Scopus (52) Google Scholar). Both eosin-succinimido-SP-Met-tRNAf and pyrene-succinimido-SP-Met-tRNAf were synthesized by the same protocol except that 3,3-dithiobis(sulfosuccinimidyl)propionate (DTSSP) was used instead of succinimide monoester of dithiodiglycolic acid. DTSSP is water-soluble and will cross-link two tRNA molecules, even though it is present at a high molar excess of the latter. The product including any DTSSP-([35S]Met-tRNA)2 that was formed was reduced by dithiothreitol. The resulting thiopropionate derivative of [35S]Met-tRNAf was then reacted with maleimides of eosin or pyrene. Cascade yellow-[35S]Met-tRNAf was synthesized by reacting the amino group of [35S]Met-tRNAfwith the succinimidyl ester of cascade yellow followed by ethanol precipitation. HPLC purification was carried out for all the modified Met-tRNA species essentially as described for coumarin-Met-tRNAf (1.Kudlicki W. Odom O.W. Kramer G. Hardesty B. J. Mol. Biol. 1994; 244: 319-331Crossref PubMed Scopus (52) Google Scholar). The elution solvents used for HPLC separation of the Met-tRNAf species were 20 mmTris acetate (pH 7.5), 10 mm magnesium acetate, 400 mm NaCl (solvent A), and 60% methanol in solvent A (solvent B). Coumarin-Met-tRNAf and eosin-Met-tRNAf were eluted from the C3 column with 50% of solvent B, whereas pyrene-Met-tRNAf eluted with 80% of solvent B. Cascade yellow-Met-tRNAf was eluted with 20% of solvent B. Propagation of the plasmids, isolation of T7 RNA polymerase, and preparation of the E. colicell-free extract (S30) were carried out as described (8.Kramer G. Kudlicki W. Hardesty B. Hames G. Higgins S. Protein Expression. Oxford University Press, Oxford, United Kingdom1999: 201-223Google Scholar). The in vitro coupled transcription/translation assay was used (9.Kudlicki W. Odom O.W. Kramer G. Hardesty B. J. Biol. Chem. 1994; 269: 16549-16553Abstract Full Text PDF PubMed Google Scholar) with some modifications in the salt concentration of the reaction mixture, which included 30 mm of (NH4)2S2O3. However, unfractionated tRNA and folinic acid were omitted. Bovine RHO and CAT were synthesized using f[35S]Met-tRNA or fluorophore-[35S]Met-tRNA (generally at 10,000 Ci/mol) as the radioactive precursors and the E. coli S30 fraction with non-linearized plasmids and T7 RNA polymerase plus rifampicin. In the absence of plasmid, trichloroacetic acid-insoluble material was less than 5% of the polypeptides formed in the presence of plasmid. These blank values were subtracted. About 40 nmol of were for 30 at in a of with of in reaction in the presence of the reaction was at for 10 to the About of this preparation was to of the transcription/translation reaction after for the at and then the at was for 10 About a of trichloroacetic acid was and the prepared for polyacrylamide gel which was carried out as described gel electrophoresis in was essentially as described by and G. Biochem. PubMed Scopus Google Scholar). the were they were to a × and in a RHO was to the of B. Chem. Google Scholar) as given by Kudlicki (9.Kudlicki W. Odom O.W. Kramer G. Hardesty B. J. Biol. Chem. 1994; 269: 16549-16553Abstract Full Text PDF PubMed Google Scholar). CAT was by the of Biochem. PubMed Scopus Google Scholar). RHO was synthesized using cascade or to initiate protein coupled transcription/translation under cascade or was as described for RHO (1.Kudlicki W. Odom O.W. Kramer G. Hardesty B. J. Mol. Biol. 1994; 244: 319-331Crossref PubMed Scopus (52) Google Scholar). containing after chromatography were used for These were carried out on a from were at using an of for cascade yellow and for pyrene. In of nascent CAT peptides were synthesized with for 30 at the ribosomal fraction was by and in 400 of 20 mm 10 30 mm and mm dithiothreitol. The was with an of the was of were to the which was then for 10 at the was Both the and the were on the of from were from the was as described in Ref. B. Odom O.W. W. PubMed Scopus Google Scholar. transcription/translation in the in vitro E. coli system a by which a modified methionine can be incorporated from a nascent This been used to fluorescent probes in size and can be incorporated at the N terminus of bovine RHO kDa, Ref. D. R. J. S. Horowitz J. Biol. Chem. Full Text PDF PubMed Google Scholar) or CAT kDa, Ref. D. PubMed Scopus Google Scholar) nascent peptides. we analyzed the modification at the N of RHO or CAT affected the to which these polypeptides were produced in to the initiation with of full-length protein from the ribosomes was by their W. Odom O.W. Kramer G. Hardesty B. J. Biol. Chem. 1994; 269: 16549-16553Abstract Full Text PDF PubMed Google Scholar). The data are in these synthesis was carried out in the presence of or The methionine can be incorporated at the N terminus of the the was for 30 to of the polypeptides initiated with the initiator data W. G. and B. showed that covalently attached to a peptide as a for and methionine In addition, that the coumarin and the derivative of methionine is chemically under the of RHO and CAT initiated with fMet-tRNAf or fluorophore-Met-tRNAf and of the × 10 × 10 or CAT was synthesized by coupled transcription/translation under optimal of of reaction with cascade or fMet-tRNAf as the initiator 30 of at an from the reaction was by trichloroacetic acid and was used to the The data are for a reaction for incorporation of the derivatives in the absence of plasmid were This was subtracted. in plasmid was from the values given for The data are the values from three in a RHO or CAT was synthesized by coupled transcription/translation under optimal of of reaction with cascade or fMet-tRNAf as the initiator 30 of at an from the reaction was by trichloroacetic acid and was used to the The data are for a reaction for incorporation of the derivatives in the absence of plasmid were This was subtracted. in plasmid was from the values given for The data are the values from three The data in indicate a reduction in incorporation of N-terminal methionine of for both RHO and CAT when peptide synthesis was initiated with fluorophore-Met-tRNAf compared with In the were the that was with the or polypeptides that were produced that full-length protein was formed and into native however, that CAT was reduced than have been from incorporation of from when synthesis was initiated with fluorophore-Met-tRNAf. by Kudlicki (1.Kudlicki W. Odom O.W. Kramer G. Hardesty B. J. Mol. Biol. 1994; 244: 319-331Crossref PubMed Scopus (52) Google Scholar) that full-length rhodanese synthesis was initiated with coumarin-Met-tRNAf coumarin at N terminus. We tested pyrene and cascade yellow can be at the N of RHO polypeptides from the ribosomes. Both pyrene and eosin derivatives of Met-tRNAf were synthesized by the same and to be Cascade yellow, on the other is a a and is linked to the α-amino group of methionine by reaction of succinimidyl coupled transcription/translation in the presence of or cascade the ribosomal and the were or cascade RHO was by gel chromatography (1.Kudlicki W. Odom O.W. Kramer G. Hardesty B. J. Mol. Biol. 1994; 244: 319-331Crossref PubMed Scopus (52) Google Scholar). are in to the fluorophore-Met-tRNAf. The cascade is to the with a of the of compared with the of the fluorophore-Met-tRNAf is at RHO with an of showed a large in compared with that this probe is in a at the N terminus of The data in that pyrene and cascade yellow are present on the which was initiated with the fluorophore-Met-tRNAf. The results in that under the the different probes were incorporated at different at the N of the two RHO and The of peptide that incorporation of for about and incorporation of at the N terminus of RHO or CAT peptides when protein synthesis was initiated with With a was the the incorporation of the probes attached covalently to the from to of the with formyl-methionine. With both plasmids containing the RHO or the CAT coding the of reduced initiation was the least and eosin-Met-tRNAf the largest effect. In G. and B. we that the of eosin-Met-tRNAf to ribosomes was reduced compared with of that in the coupled transcription/translation system used synthesis after the for about 20 after which the rate and after 30 of at W. Odom O.W. Kramer G. Hardesty B. Horowitz J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). In the to the data in was to 30 to of initiated peptides. The data given in indicate a reduction in the amount of RHO or CAT, when formed the N terminus. RHO, this reduction is with the data in and may be by the in the rate of initiation in this However, with CAT, a in to the reduced incorporation of the was we to the was affected after nascent peptides been initiated with this we analyzed the of nascent polypeptides with the results given in the The of synthesized polypeptides of the two initiated with fMet-tRNAf or of the of fluorophore-Met-tRNAf was after followed by as described under The of peptides from in the initiator tRNA are for RHO and CAT in In of RHO full-length protein is although to different is the with all initiator tRNA In addition, a of peptide is These are in the size of kDa, the of full-length These different at which translation is or The of this pause in translation not been of was to modification of the N terminus of the by different fluorophores the of these peptides. This not to be the at least on the The results given in for RHO the reduction in synthesis of polypeptides in for Synthesis of was the The same is for CAT In addition, with CAT, is a reduction in full-length product formed when synthesis was initiated with all fluorophore-Met-tRNAf. CAT is reduced on the of polypeptides of and with the CAT peptides having a of at the N terminus of CAT the effect. of these peptides was in the of CAT synthesis in CAT synthesis was initiated with fMet-tRNAf A), polypeptides in the size of about 4 were after and 10 of but were reduced after With cascade or pyrene-Met-tRNAf these low molecular polypeptides of amino in the after 10 and 30 of as by after gel The results with RHO were different the of the to be full-length rhodanese cascade or formed the N terminus. In with at the N the results major in the of RHO nascent peptides during the of and full-length RHO is efficiently formed. In the of CAT, however, the rate of to be reduced when the N terminus. The results that fluorophores of different size and on ribosomal pausing at pause sites when CAT is These in accumulation of peptides be that the peptide in from the radioactive methionine which different fluorophores are covalently at the N terminus of the that these polypeptides were not by of or were the of from the we carried out in which the reaction after coupled transcription/translation the presence of or pyrene-Met-tRNAf and amino was with The are given under The polypeptides were analyzed by SDS-PAGE and These results are given in was that of peptides in reacted with were when the other fluorophore-Met-tRNAf species initiated peptide synthesis not The results indicate that the low molecular CAT peptides are not and peptides but are nascent peptides to the ribosomal Fluorescence were to the of the N-terminal probe on nascent CAT peptides. With a in in the of the and in will indicate with other or in the environment. CAT peptides were synthesized with pyrene-Met at their N terminus under in which low molecular weight nascent peptides on the and then the ribosomal fraction was and to the of the ribosomes nascent pyrene-CAT peptides. This fraction was then with and analyzed in the The after reaction is by in A large in was This in a of the probe to a environment. for the in as as are in in this are the values for in of CAT and from ribosomes by fraction with pyrene-CAT fraction after fraction the pyrene given were derived from the for as in of in a The given were derived from the for as in of the measurements in were the was to the peptides were from the ribosomes. Both the ribosomes and the resulting were to measurements with the results in The results indicate that the pyrene-CAT peptides were into the The data for and included in We these results to indicate a in the of the pyrene at the N terminus of the nascent CAT peptides from a less to a We the less hydrophilic the of the nascent peptides on the ribosomes is than the of pyrene-Met-tRNAf we the results to indicate a relatively hydrophilic of the probe when the pyrene-CAT peptides are to the ribosomes inside the tunnel of the 50 S ribosomal subunit as discussed the peptides may with the pyrene inside the peptide in a The fluorescent probes used in the present in their molecular and of the dimensions of the probes that the fluorophores used from to 20 in and about in with the of eosin, which a of about 11 with 4 to the system. All fluorophore-Met-tRNAf tested a reduction in both CAT and RHO polypeptides which in the of RHO can be to less the effect. In addition, the results above indicate that translational pausing at some of the pause sites is when probes are incorporated at the N terminus of caused a in pausing than coumarin in CAT nascent peptides of about amino in A effect than with coumarin is with cascade yellow, which is in The pyrene derivative to be the on the HPLC elution of the of the fluorophores although it is in a size to cascade yellow and coumarin. The results indicate that the character of the probe is the of pausing at specific Low molecular weight peptides of CAT were previously when protein synthesis was initiated with coumarin-Met-tRNAf and was by the amount of the E. coli extract used in the assay (5.Tsalkova T. Kramer G. Hardesty B. J. Mol. Biol. 1999; 286: 71-81Crossref PubMed Scopus (15) Google Scholar). was that the amino acid sequence at the N terminus be the in the accumulation of peptides. CAT with a hydrophilic N terminus was to be affected by the derivative of N-terminal during the required folding of the nascent peptide to for through the tunnel of the large ribosomal subunit (5.Tsalkova T. Kramer G. Hardesty B. J. Mol. Biol. 1999; 286: 71-81Crossref PubMed Scopus (15) Google Scholar). We to this as above on the of RHO and CAT were carried out with E. coli N-terminal amino acid sequence S. A. PubMed Scopus Google Scholar) a J. J. Mol. Biol. PubMed Scopus Google Scholar) to however, was accumulation of low molecular weight nascent when synthesis was initiated with or cascade yellow-Met-tRNAf not causes CAT peptides to when pyrene, cascade yellow, or coumarin the N be out that an N-terminal probe on nascent CAT peptides to specific when a of was The values for RHO and for protein were and kDa, T. Odom O.W. Kramer G. Hardesty B. J. Mol. Biol. 1998; 278: 713-723Crossref PubMed Scopus (35) Google Scholar). The nascent CAT peptides inside the ribosomal tunnel may a which is affected by the bulky N-terminal in to the structures by the other The structure of CAT J. Mol. Biol. 1990; PubMed Scopus Google Scholar) is with of as is the with RHO G. J. PubMed Scopus Google Scholar). The structure of is not structure to and B. J. PubMed Scopus Google Scholar) an structure for the first 40 amino of The N terminus of RHO is then an is formed by amino The structure of CAT an N-terminal followed by a is formed. We not these in the N-terminal structures are the for the when or cascade yellow-Met are at the N terminus of the nascent peptides. We this be the from the results above is that the N-terminal pyrene on the nascent CAT peptides is in a relatively hydrophilic environment. The CAT peptides are in the of 4 about the the N terminus to The recent of the S from of A. R. R. J. J. Mol. Biol. 1998; PubMed Scopus Google Scholar) a tunnel through the 50 S subunit. The at a of the of tRNA at the of the which is to be the for the nascent We estimated the of the tunnel to be about and and Å, on these data with recently data on the 50 S subunit B. R. R. J. T. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). these with the structure for the N-terminal 40 amino of the CAT the question the N-terminal pyrene is within the data R. 1998; PubMed Scopus Google Scholar) obtained with an N-terminal probe on different nascent polypeptides cross-linking to specific nucleotides of the 23 S RNA. the synthesized peptides a of about 30 amino cross-linking to nucleotides of the ribosomal RNA was all three polypeptides of this and gene cross-linking to nucleotides and was additional were with the These results are they that different peptides of the same different is in the This finding that nascent peptides are not the ribosomal tunnel in a the results a hydrophilic that the nascent peptide may inside the ribosomal results with nascent pyrene-CAT peptides this We the gift of cytidylic acid-puromycin from Dr. (Department of Molecular and We (Department of and Biochemistry, University of for the dimensions of the probes by Molecular In addition, we for and for with the