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

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.

Initiation of protein synthesis by a specific initiator tRNA Met at an AUG codon is a universally conserved step in gene expression for both eukaryotes and prokaryotes. In prokaryotes, methionine linked to the initiator tRNA Met is formylated to give fMet-tRNA f . 1 However, peptide initiation can be carried out with other N-acyl derivatives of Met-tRNA f , many of which can be synthesized chemically from Met-tRNA f .
We have synthesized coumarin maleimide-S-acetyl-Met-tRNA f 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)(2)(3)(4). 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) 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 ␣-amino group of methionine on the initiator-tRNA as efficiently as with fMet-tRNA f ? Initiation of protein synthesis is usually the rate-controlling step and a major point for regulation of translation (6). 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), 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 (EC 2.3.1.28) was initiated in the presence of fluorophore-Met-tRNA f . 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-tRNA f , the translational machinery worked less efficiently. With all fluorophore-Met-tRNA f 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.

Methods
Synthesis of Fluorophore-Met-tRNA f Species-Coumarin-SAc-Met-tRNA f was prepared as described previously (1). Both eosin-succinimido-SP-Met-tRNA f and pyrene-succinimido-SP-Met-tRNA f 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-([ 35 S]Met-tRNA) 2 that was formed was reduced by dithiothreitol. The resulting thiopropionate derivative of [ 35 S]Met-tRNA f was then reacted with maleimides of eosin or pyrene.
Cascade yellow-[ 35 S]Met-tRNA f was synthesized by reacting the amino group of [ 35 S]Met-tRNA f with 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-tRNA f (1). The elution solvents used for HPLC separation of the Met-tRNA f species were 20 mM Tris acetate (pH 7.5), 10 mM magnesium acetate, 400 mM NaCl (solvent A), and 60% methanol in solvent A (solvent B). Coumarin-Met-tRNA f and eosin-Met-tRNA f were eluted from the C3 column with 50% of solvent B, whereas pyrene-Met-tRNA f eluted with 80% of solvent B. Cascade yellow-Met-tRNA f was eluted with 20% of solvent B.
The Cell-free System-Propagation of the plasmids, isolation of T7 RNA polymerase, and preparation of the E. coli cell-free extract (S30) were carried out as described (8). The in vitro coupled transcription/ translation assay was used (9) with some modifications in the salt concentration of the reaction mixture, which included 30 mM of (NH 4 ) 2 S 2 O 3 . However, unfractionated tRNA and folinic acid were omitted. Bovine RHO and CAT were synthesized using f[ 35 S]Met-tRNA or fluorophore-[ 35 S]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.
Radioactive Labeling of C-puro and Its Reaction with Nascent Polypeptides on the Ribosomes-About 40 nmol of C-puro were incubated for 30 min at 37°C in a total volume of 500 l with 150 units of T4 polynucleotide kinase (Life Technologies, Inc.) in "forward" reaction buffer in the presence of 0.2 mM [␥-32 P]ATP, 5000 Ci/mol. Then, the reaction mixture was held at 65°C for 10 min to inactivate the enzyme. About 1.5 l of this preparation was added to each 15 l of the transcription/translation reaction mixture after its incubation for the indicated time at 37°C, and then the incubation at 37°C was continued for 10 min. About a 7-fold volume of 7% cold trichloroacetic acid was added and the sample prepared for polyacrylamide gel electrophoresis, which was carried out as described below.
Gel Electrophoresis and Enzyme Assays-Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in Tricine was performed essentially as described by Schä gger and von Jagow (10). After the gels were dried, they were exposed to a phosphor screen (35 ϫ 43 cm) and scanned in a PhosphorImager (Molecular Dynamics).
RHO activity was assayed according to the method of Sörbo (11) as given by Kudlicki et al. (9). CAT enzyme activity was assayed by the method of Sleigh (12).
Sample Preparation for Fluorescence Measurements-RHO was synthesized using cascade yellow-or pyrene[ 35 S]Met-tRNA f to initiate protein synthesis. After coupled transcription/translation under standard conditions, cascade yellow-RHO or pyrene-RHO was purified as described for coumarin-labeled RHO (1). Fractions containing fluorophore-RHO after Sephadex G100 chromatography were used for fluorescence measurements. These were carried out on a model 8000C photon counting spectrofluorometer from SLM-Aminco (Urbana, IL.). Emission spectra were recorded at 1 nm intervals using an excitation wavelength of 408 nm for cascade yellow and 338 nm for pyrene.
In another set of experiments, nascent CAT peptides were synthesized with pyrene-[ 35 S]Met-tRNA f for 30 min at 37°C. Then, the ribosomal fraction was isolated by Airfuge (Beckman) centrifugation and resuspended in 400 l of 20 mM Tris-HCl, pH 7.5, 10 mM Mg(OAc) 2 , 30 mM (NH 4 OAc), and 1 mM dithiothreitol. The fluorescence emission spectrum was recorded with an excitation wavelength of 338 nm. After the spectrum was taken, 6 l of [ 32 P]C-puro were added to the sample, which was then incubated for 10 min at 37°C before the emission spectrum was recorded again. Both the spectrum and the relative quantum yield were normalized on the basis of radioactivity from [ 35 S]Met. Relative quantum yields were determined from the integrated emission spectra. Anisotropy was calculated as described in Ref. 13.

Incorporation of Coumarin, Cascade Yellow, Eosin, and Pyrene Derivatives of Methionine at the N Terminus of Polypep-
tides-Coupled transcription/translation in the in vitro E. coli system provides a method by which a modified methionine can be incorporated from derivatized Met-tRNA f into a nascent peptide. This approach has been used here to study whether fluorescent probes differing in size and charge can be incorporated at the N terminus of bovine RHO (33 kDa, Ref. 14) or CAT (25.6 kDa, Ref. 15) nascent peptides.
First, we analyzed whether the fluorophore-Met modification at the N termini of RHO or CAT affected the extent to which these polypeptides were produced in comparison to the initiation with fMet-tRNA f . Production of full-length protein released from the ribosomes was monitored by their enzymatic activity (cf. Ref. 9). The data are presented in Table I. For these experiments, polypeptide synthesis was carried out in the presence of f[ 35 S]Met-tRNA f or fluorophore-[ 35 S]Met-tRNA f . The radioactively labeled methionine can be incorporated only at the N terminus of the proteins; the incubation was continued for 30 min to achieve maximal extension of the polypeptides initiated with the labeled initiator tRNA. Previous unpublished data 2 showed that coumarin-Met covalently attached to a peptide cannot serve as a substrate for deformylase and methionine aminopeptidase. In addition, earlier unpublished observa- tions 2 indicated that the thioether bond between coumarin and the thiol derivative of methionine is chemically stable under the incubation conditions used. The data in Table I indicate a reduction in incorporation of radioactively labeled N-terminal methionine of 40 -60% for both RHO and CAT when peptide synthesis was initiated with fluorophore-Met-tRNA f compared with fMet-tRNA f . In parallel, the measured enzymatic activities were reduced; yet, the fact that enzymatic activity was associated with the fluorophore-Met-RHO or -CAT polypeptides that were produced indicates that apparently full-length protein was formed and folded into its native structure. Note, however, that CAT activity was reduced more strongly than would have been expected from incorporation of radioactivity from Met when synthesis was initiated with fluorophore-Met-tRNA f .
Earlier observation by Kudlicki et al. (1) indicated that fulllength rhodanese whose synthesis was initiated with coumarin-Met-tRNA f contained coumarin at its N terminus. We tested whether pyrene and cascade yellow can be detected at the N termini of RHO polypeptides released from the ribosomes. Both pyrene and eosin derivatives of Met-tRNA f were synthesized by the same reactions and offer to be equally stable. Cascade yellow, on the other hand, is a zwitterion, has a more flexible structure, and is linked to the ␣-amino group of methionine by reaction of its succinimidyl ester.
After coupled transcription/translation in the presence of pyrene-or cascade yellow-Met-tRNA f, the ribosomal and the supernatant fractions were separated. Released pyrene-or cascade yellow-labeled RHO was purified by gel filtration chromatography (1). Their fluorescence spectra are shown in Fig. 1 in comparison to the respective fluorophore-Met-tRNA f . The cascade yellow-RHO spectrum (panel A) is shifted to the blue with a maximum of the emission spectrum of 518 nm compared with the spectrum of the fluorophore-Met-tRNA f whose emission maximum is at 542 nm. Pyrene-labeled RHO (panel B) with an emission maximum of 398 nm showed a large increase in quantum yield compared with pyrene-Met-tRNA f , indicating that this environmentally sensitive probe is in a more hydrophobic surrounding at the N terminus of rhodanese. The data presented in Fig. 1 demonstrate that pyrene and cascade yellow are indeed present on the protein, which was initiated with the respective fluorophore-Met-tRNA f .
The Rate of Initiation Is Affected When RHO or CAT Syntheses Are Initiated with Fluorophore-Met-tRNA f Compared with fMet-tRNA f -The results shown in Fig. 2 demonstrate that under the conditions used, the structurally different probes were incorporated at different initial rates at the N termini of the two proteins tested, RHO and CAT. The time course of peptide formation indicates that nearly linear incorporation of [ 3S S]Met proceeded for about 3-5 min and highest incorporation of [ 35 S]Met at the N terminus of RHO or CAT peptides occurred when protein synthesis was initiated with f[ 35 S]Met-tRNA f . With fluorophore-Met-tRNA f , a brief lag period was observed. At the 5-min time point, the percentage incorporation of the various probes attached covalently to the [ 35 S]methionine ranged from 30% to 62% of the level reached with formylmethionine. With both plasmids containing either the RHO or the CAT coding sequence, the order of reduced initiation was identical; pyrene-Met-tRNA f had the least and eosin-Met-tRNA f had the largest effect. In unpublished experiments, 3 we noted that the binding of eosin-Met-tRNA f to salt-washed ribosomes was reduced compared with binding of fMet-tRNA f .
Earlier observations indicated that in the coupled transcrip-tion/translation system used here, polypeptide synthesis (mainly elongation after the initial 5 min) proceeds nearly linearly for about 20 min, after which the rate declines rapidly and approaches zero after 30 min of incubation at 37°C (16). In the experiments leading to the data presented in Table I, incubation time was extended to 30 min to allow maximum elongation of initiated peptides. The data given in Table I indicate a reduction in the amount of enzymatically active RHO or CAT, when fluorophore-Met formed the N terminus. For RHO, this reduction is compatible with the data presented in Fig. 3 and may be explained by the differences in the rate of initiation shown in this figure. However, with CAT, a larger decrease in enzymatic activity relative to the reduced incorporation of the fluorophore was measured. Therefore, we wanted to determine whether the elongation was affected after nascent peptides had been initiated with fluorophore-Met. For this reason, we analyzed the pattern of nascent polypeptides with the results given in the following section. The Pattern of RHO and CAT Pause-site Peptides-The pattern of synthesized polypeptides of the two test proteins initiated with fMet-tRNA f or each of the four forms of fluorophore-Met-tRNA f was revealed after SDS-PAGE, followed by phosphorimaging, as described under "Experimental Procedures." The patterns of peptides radioactively labeled from [ 35 S]Met in the initiator tRNA are shown for RHO and CAT in Fig. 3. In case of RHO (lanes 1-5), full-length protein is formed, although to different extent. It is the most prominent band with all initiator tRNA species. In addition, a ladder of discrete peptide bands is seen. These "pause-site peptides" are in the size range of 5-33 kDa, the mass of full-length RHO. These different bands represent points at which translation is temporarily slowed or stopped. The cause of this pause in translation has not been established. One of our objectives was to determine if modification of the N terminus of the test proteins by attaching different fluorophores affects the pattern of these pause-site peptides. This does not seem to be the case, at least on the qualitative level.
The results given in Fig. 3 for RHO confirm the reduction in overall synthesis of polypeptides presented in Table I for fluorophore-Met-RHO. Synthesis of eosin-RHO was the lowest. The same is true for CAT (Fig. 3, lane 10). In addition, with CAT, there is a remarkable reduction in full-length product formed when synthesis was initiated with all fluorophore-Met-tRNA f . Full-length CAT is reduced on the expense of small polypeptides of 6.5 kDa and below, with the most prominent CAT peptides having a mass of 3.5-4 kDa (Fig. 3, lanes 7-10). Pyrene-Met at the N terminus of CAT exerts the strongest effect. The results with RHO were completely different (Fig. 5). After the initial 3 min of incubation, the most prominent polypeptide appeared to be full-length rhodanese regardless whether fMet, cascade yellow-Met, pyrene-Met, or eosin-Met formed the N terminus.
In summary, comparing fMet with fluorophore-Met at the N terminus, the results show no major differences in the pattern of transient RHO nascent peptides during the time of elongation, and full-length RHO is efficiently formed. In the case of CAT, however, the rate of elongation appears to be reduced when fluorophore-Met forms the N terminus. The results demonstrate that fluorophores of different size and hydrophobicity cause quantitative effects on ribosomal pausing at pre-existing pause sites when CAT mRNA is translated. These effects result in accumulation of fluorophore-Met-CAT peptides below 6 kDa.
It should be emphasized that the peptide patterns shown in Figs. 3-5 result from the radioactive methionine (to which different fluorophores are covalently attached) at the N terminus of the polypeptides. To demonstrate that these polypeptides were not generated by proteolytic degradation of larger products or were the result of premature release from the ribosomes, we carried out experiments in which the reaction mixture after coupled transcription/translation (in the presence of non-radioactive fMet-or pyrene-Met-tRNA f and nonradioactive amino acids) was incubated with [ 32 P]C-puro. The experimental details are given under "Experimental Procedures." The radioactively labeled polypeptides were analyzed by SDS-PAGE and phosphorimaging. These results are given in Fig. 6. It was observed that most sizes of pause-site peptides seen in Fig. 3-5 reacted with puromycin. Similar [ 32 P]C-puropeptide patterns were observed when the other fluorophore-Met-tRNA f species initiated peptide synthesis (data not presented). The results indicate that the low molecular mass CAT peptides are not aborted and released peptides but are nascent peptides bound to the ribosomal P site.
Nascent CAT Peptides Are in a Relatively Hydrophilic Environment-Fluorescence techniques were applied to study the environment of the N-terminal probe on nascent CAT peptides. With a sensitive probe, changes in quantum yield, in the maximum of the emission spectrum, and in anisotropy will indicate interaction with other molecules or changes in the environment.
CAT peptides were synthesized with pyrene-Met at their N terminus under conditions in which low molecular weight nascent peptides accumulate on the ribosomes, and then the ribosomal fraction was isolated (see "Experimental Procedures") and subjected to fluorescence measurements. Fig. 7 (spectrum 1) shows the fluorescence emission spectrum of the resuspended ribosomes bearing nascent pyrene-CAT peptides. This fraction was then treated with C-puro (see "Experimental Procedures") and analyzed again in the fluorometer. The spectrum after puromycin reaction is shown by line 2 in Fig. 7. A large increase in fluorescence intensity was observed. This increase in quantum yield signals a move of the probe to a much more hydrophobic environment. Calculated fluorescence parameters for the spectra shown in Fig. 7 as well as anisotropy calculations are compiled in Table II. Included in this table are the corresponding values for pyrene-Met-tRNA f free in buffered solution.
After the measurements indicated in Fig. 7 (spectrum 2) were completed, the sample was centrifuged to analyze whether the pyrene-CAT-C-puro peptides were separated from the ribosomes. Both fractions, the resuspended ribosomes and the resulting supernatant, were subjected to fluorescence measurements with the results shown in Fig. 7 (spectra 3 and 4,  respectively). The results indicate that the pyrene-CAT peptides were released into the soluble fraction. The quantitative fluorescence data for spectra 3 and 4 are included in Table II. We interpret these results to indicate a change in the environment of the pyrene moiety at the N terminus of the nascent CAT peptides from a less hydrophobic (relatively hydrophilic) to a very hydrophobic surrounding. We emphasize the less hydrophobic/relatively hydrophilic aspect, because the relative fluorescence quantum yield of the nascent pyrene-Met-CAT peptides on the ribosomes is appreciably higher than the quantum yield of free pyrene-Met-tRNA f (Table II). Further, we interpret the results to indicate a relatively hydrophilic environment of the probe when the pyrene-CAT peptides are bound to the ribosomes probably inside the tunnel of the 50 S ribosomal subunit as discussed below. When released, the peptides may collapse with the pyrene buried inside the peptide in a very hydrophobic pocket. DISCUSSION The fluorescent probes used in the present study differ in their molecular size, shape, charge, and hydrophobicity. Com  4). Synthesis was carried out with 5 l of S30 in the reaction mixture during an incubation at 37°C for 30 min. Immediately after this incubation, [ 32 P]C-puro was added to the reaction mixtures and the incubation was continued as described under "Experimental Procedures." The reaction products were analyzed by SDS-PAGE and visualized by phosphorimaging.

FIG. 7.
Fluorescence spectra of ribosome-bound pyrene-labeled CAT peptides and their release by puromycin. Pyrenelabeled CAT was synthesized in the cell-free bacterial system and after 30 min of incubation, the ribosomal fraction was obtained by Airfuge centrifugation. The fluorescence spectrum of the resuspended ribosomes bearing 4 pmol of CAT peptides was taken (spectrum 1). Then, 6 l of C-puro was added to the same sample, which was incubated for 10 min at 37°C, followed by fluorescence measurements (spectrum 2). The ribosomes were separated by a second Airfuge centrifugation and fluorescence spectra were recorded for both fractions, the resuspended ribosomes (spectrum 3) and the supernatant fraction (spectrum 4). Based on radioactivity, about 50% of the puromycin-tagged CAT peptides were released. The spectrum of 5 pmol of free pyrene-Met-tRNA f is shown as comparison (spectrum 5).

TABLE II
Fluorescence properties of pyrene-labeled CAT and its release from ribosomes by puromycin The fluorescence parameters given here were derived from the spectra for samples 1-4 as shown in Fig. 7. E max , wavelength of emission maximum; A, fluorescence anisotropy; Rel Q, relative fluorescence quantum yield. which has a diameter of about 11 Å with 4 bromine atoms bonded to the xanthen ring system. All fluorophore-Met-tRNA f molecules tested cause a reduction in both CAT and RHO polypeptides formed, which in the case of RHO can be traced to less efficient initiation. Eosin-Met-tRNA f has the most pronounced effect. In addition, the results presented above indicate that translational pausing at some of the early pause sites is prominent when probes are incorporated at the N terminus of CAT. Pyrene caused a greater increase in pausing than coumarin in producing CAT nascent peptides of about 30 -35 amino acids in length. A stronger effect than with coumarin is also observed with cascade yellow, which is zwitterionic in nature. The pyrene derivative appears to be the most hydrophobic (based on the HPLC elution profile of the fluorophore-Met-tRNA) of the fluorophores tested, although it is in a similar size range to cascade yellow and coumarin. The results indicate that neither hydrophobicity nor the ionic character of the probe is the unique cause of pausing at specific sites.
Low molecular weight pause-site peptides of CAT were observed previously when protein synthesis was initiated with coumarin-Met-tRNA f and elongation was slowed by reducing the amount of the E. coli extract used in the assay (5). It was suggested that the primary amino acid sequence at the N terminus might be the determining element in the accumulation of pause-site peptides. CAT with a very hydrophilic N terminus was assumed to be strongly affected by the hydrophobic derivative of N-terminal Met during the required folding of the nascent peptide to allow for its path through the tunnel of the large ribosomal subunit (5). We wish to modify this hypothesis, because similar experiments as reported above on the elongation of RHO and CAT were carried out with another protein, E. coli release factor RF-1. Its N-terminal amino acid sequence (17) gives a hydrophobicity plot (18) very similar to CAT; however, there was no accumulation of low molecular weight nascent peptides, when RF-1 synthesis was initiated with pyrene-or cascade yellow-Met-tRNA f (data not presented).
What causes CAT peptides to accumulate when pyrene, cascade yellow, or coumarin forms the N terminus? It should be pointed out that an N-terminal probe on nascent CAT peptides became accessible to specific antibodies only when a mass of 8.5 kDa was reached. The corresponding values for RHO and for MS2 coat protein were 6 and 4.5 kDa, respectively (4). The nascent CAT peptides inside the ribosomal tunnel may form a more compact structure, which is affected by the bulky Nterminal extension in contrast to the secondary structures acquired by the other proteins tested. The crystal structure of monomeric CAT (19) is very compact with no formation of domains as is the case with RHO (20). The crystal structure of RF-1 is not known yet. Secondary structure prediction according to Garnier and Robson (21) indicates an ␣-helical structure for the first 40 amino acids of RF-1. The N terminus of RHO is quite flexible; then an ␣-helix is formed by amino acids 11-22. The crystal structure of CAT indicates an N-terminal ␤-sheet (amino acids 1-12), followed by a short ␣-helix before another ␤-sheet is formed. We do not know whether these differences in the N-terminal secondary structures are the cause for the effects seen when pyrene-or cascade yellow-Met are at the N terminus of the nascent peptides. We assume this might be the case.
An important conclusion from the results presented above is that the N-terminal pyrene on the nascent CAT peptides is in a relatively hydrophilic environment. The most prominent CAT peptides are in the range of 4 kDa: about half the mass before the N terminus becomes accessible to antibodies. The recent model of the 70 S ribosome developed from image reconstruction of electron micrographs (22) confirms a structured tunnel through the 50 S subunit. The images at a resolution of 15 Å position the CCA end of P-site bound tRNA at the mouth of the tunnel, which is assumed to be the conduit for the nascent polypeptides. We estimated the length of the tunnel to be about 70 Å and its width between 16 and 19 Å, based on these cryoelectron microscopy data together with recently published crystallographic data on the 50 S subunit (23). Combining these numbers with the secondary structure for the N-terminal 40 amino acids of the CAT still leaves the question open where the N-terminal pyrene is localized within the tunnel. Recent data (24) obtained with an N-terminal photo-activatable probe on different nascent polypeptides indicated cross-linking to specific nucleotides of the 23 S RNA. When the synthesized peptides had a length of about 30 amino acids, cross-linking to several nucleotides of the ribosomal RNA was observed. For all three polypeptides of this length (ompA peptide, tetracycline resistant gene-peptide, and T4 gene 60 peptide), cross-linking to nucleotides 2062 and nucleotide 2609 was found consistently; additional cross-links were determined with the second peptide. These results are quite remarkable; they show that different peptides of the same length gave different cross-links. Furthermore, nucleotide 2609 is in domain V, the peptidyltransferase center. This finding indicates that nascent peptides are not traversing the ribosomal tunnel in a linear arrangement. Most importantly, the results suggest a hydrophilic environment that the nascent peptide may encounter inside the ribosomal tunnel. Our fluorescence results with nascent pyrene-CAT peptides support this notion.