INTRODUCTION

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Bacteriophage T4 gene 60 mRNA, which encodes a subunit of the type II T4 DNA topoisomerase, contains a 50-nucleotide (nt) coding gap between codons 46 and 47 that is bypassed by ribosomes in order to synthesize the full-length protein (10). The cis-acting recoding signals required for this unusual event have been studied in detail (23). These signals are depicted in Fig. 1 and include (i) matching GGA codons bordering the gap (referred to as takeoff and landing sites), (ii) a stop codon immediately after the takeoff site, (iii) a short stem-loop structure in the 5' end of the coding gap, (iv) an optimal 50-nt spacing, and (v) a region of the nascent peptide translated from codons preceding the gap.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Efficient T4 gene 60 translational bypassing requires five bypassing signals. These signals include matching GGA codons bordering the gap, a stop codon, a short stem-loop structure, an optimal 50-nt spacing, and a region of the nascent peptide.

Three steps describe the proposed mechanism of bypassing. The first involves dissociation of the codon-anticodon pairing between the peptidyl tRNA₂^Gly and the GGA at the takeoff site. The second is movement of the mRNA through the ribosome, which brings the landing site to the peptidyl tRNA. The third occurs when pairing is reestablished at the second GGA and coding resumes.

The critical part of the nascent peptide is amino acids 17 to 34, although positions on either side of this region are also important (23). It is unclear whether this signal exerts its effect within the peptide exit channel of the ribosome or in some other location. Although the spacing between the sequence encoding the critical region of the nascent peptide and the start of the coding gap is known to be important (23), the spacing between the amino terminus and the critical region of the nascent peptide has not been analyzed, nor has the importance of the identity of the amino terminus.

The efficiency of gene 60 bypassing has been determined in vivo with gene 60-lacZ reporter constructs driven by the tac promoter. In these experiments, efficiency was determined by comparing test constructs that contained 5' fragments of gene 60 (including the coding gap) to constructs with a precise deletion of the coding gap (gap deletion). The first estimate of efficiency determined by Huang and coworkers was 70% (10). These constructs carried gene 60 sequence extending 24 codons downstream of the landing site. Weiss et al. demonstrated that similar constructs, carrying gene 60 sequence extending either 24 codons or 5 nt downstream of the landing site, gave efficiencies of 94 or 98%, respectively (23). This estimate is consistent with the inability to detect product due to termination at the stop codon following codon 46 (11). However, lability of the 46-amino-acid peptide may also account for this absence. Here, we reexamine the efficiency of bypassing and investigate the importance of the N terminus to the function of the nascent peptide signal.

	MATERIALS AND METHODS

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Bacteria. Escherichia coli K-12 SU1675 {ara (pro-lac) recA56 thi F' [proAB⁺ lacI^q]} is a recA derivative of CSH26 (22) and was used as a host strain in all experiments except those with the phoA fusions, where DH5 (phoA) (16) was used.

Plasmids. All changes in the following constructs were verified by dideoxy sequencing as described previously (22). A variant with a precise excision of the coding gap was made in parallel with each test vector.

(i) Construction of gene 60-lacZ fusions. The parent vector (4p101) used in the construction of the following lacZ fusions has been described previously (22). It is a pBR322-based vector that allows gene fusions to be made to the fifth codon of lacZ with unique HindIII and ApaI sites. A synthetic Shine-Dalgarno (SD) sequence, based on the lpp gene from E. coli, is cloned between the HindIII site and an upstream XbaI site. Translation begins at an AUG codon contained within this insert and results in the addition of 4 amino acids to the amino terminus of the nascent peptide of the downstream fusion. A tac promoter on a 269-bp fragment from pKK223-3, cloned into a BamHI site just upstream of the XbaI site, directs transcription of the gene fusion (4). Derivatives of this vector carrying fragments of gene 60 between the HindIII and ApaI sites have previously been used to estimate bypassing efficiency (10, 23). One of these, BA3 (23), was recreated by cloning an insert composed of four overlapping DNA oligonucleotides into the HindIII and ApaI sites of 4p101. This derivative was designated BX3*.

(ii) Construction of gene 60-phoA fusions. To make gene 60-phoA fusions, gene 60 sequence, from the initiation codon to 24 codons after the landing site, was amplified by PCR with oligonucleotides containing embedded NheI sites, digested with NheI, and cloned into pCGV1 (8). The forward primer includes sequence required to complete the bla signal peptide present in the vector. This inclusion ensures that the fusions are exported to the periplasm. Expression is driven by the pR and pL promoters of lambda phage and controlled by the thermosensitive repressor cI857, which is present on the vector.

(iii) Construction of gene 60 and GST-gene 60 plasmids. To construct gene 60 expression plasmids, under the control of the tac promoter, the entire coding sequence of gene 60 plus an additional 45 nt upstream of the start codon was amplified by PCR from pT60.32 (10) with oligonucleotides containing embedded SmaI and HindIII sites, digested with SmaI and HindIII, and cloned into pKK223-3 (4). To construct GST-gene 60 expression plasmids, the coding sequence of gene 60 was amplified from pT60.32 with oligonucleotides containing embedded BamHI sites, digested with BamHI, and cloned into pGEX2-T in frame with GST (19). Exact gap deletion variants of these clones were constructed by amplifying DNA from a version of pT60.32 that already contained the coding gap deletion (1).

-Gal activity assays. Whole-cell assays were based on the procedure of Miller (14), and data obtained are expressed as -galactosidase (-Gal) units. Overnight cultures of constructs with the consensus promoter (Table 1) were diluted 1:100 in Luria broth (LB) plus 2 mM IPTG (isoproply--D-thiogalactopyranoside), grown at 37°C with rapid shaking, and chilled to 0°C when they had reached an optical density at 600 nm of 0.4 to 0.8. Overnight cultures of constructs with the tac promoter (Table 1) were diluted 1:50 in LB and were induced after 1 h of growth with 2 mM IPTG for an additional 1.5 h. Assays were done at 30°C in a total volume of 1 ml of Z buffer plus cell suspension (14). The substrate was added as 0.2 ml of 4-mg/ml O-nitrophenol--galactoside, and the reaction was stopped by the addition of 0.5 ml of 1 M Na₂CO₃. All assays were performed in duplicate with at least two independent clones on at least two separate occasions. The absolute -Gal values and resulting bypassing efficiency estimates along with standard deviations are given in Table 1.

Phosphatase assays. Gene 60-phoA fusions were partially derepressed at 39°C in DH5 (16). The expression of protein was estimated with the standard colorimetric phosphatase assay (3).

In vivo pulse-chase experiments. All pulse-chase experiments were done in duplicate with at least two independent clones on at least two separate occasions.

(i) Bypass efficiency determination. Overnight cultures were grown in VBMAM-AM medium (0.5% glucose; 0.1% Difco methionine assay medium; 60 µg [each] of isoleucine, leucine, and valine per ml; 8 mM NaOH; 1× E salts [20a]; 50 µg of ampicillin per ml [5a]) and diluted 1:20 in 1 ml of the same. After a 2-h incubation at 37°C, the cultures were induced with 2 mM IPTG for 8 min. The cells were pulsed with 7.5 µCi of [³⁵S]methionine in 100 µl of VBMAM-AM for 30 s, chased with 100 µl of 4% cold methionine for 3 min, chilled on ice, and harvested by centrifugation. The cells were resuspended in 50 µl of cracking buffer (6 mol of urea per liter, and 1% sodium dodecyl sulfate [SDS] in 1× stacking buffer at Tris [pH 7.2]) (2), boiled 5 min, combined with 50 µl of 1× SDS loading buffer (17), and boiled an additional 5 min before 10-µl aliquots were loaded onto a Tris-glycine-SDS-13% polyacylamide gel. The resulting data were visualized with a Molecular Dynamics PhosphorImager. To control for loading differences, the amount of labeled gene 60 product was compared to that of the total labeled protein per lane. The resulting percentage was then used to estimate bypassing efficiency. Full-length GST-gene 60 fusions have 0.044 methionine per amino acid; the product due to termination following codon 46 has 0.0478 methionine per amino acid. This difference was used to correct the amount of the full-length test product before we calculated the final ratio.

(ii) Functional message stability determination. The basic procedure outlined above was performed with the following modifications. Overnight VBMAM-AM cultures were diluted 1:20 in 7 ml of the same medium. Following 2 h of incubation, the samples were induced with 2 mM IPTG for 8 min. At 8 min, 0.5-ml aliquots of culture were transferred to separate tubes containing 50 µl of labeling mix, where they were chased as described above with 50 µl of 4% cold methionine. Rifampin (500 µg/ml) was added to the remaining cultures, and 0.5-ml aliquots were removed and assayed after 1, 2, 3.5, 5, and 10 min. The amount of labeled gene 60 product per lane was determined for three independent clones. To correct for loading differences among the clones, the result for each clone was multiplied by the ratio of the amount of total labeled protein in its lane to the average amount of total labeled protein in the lanes of all three clones. The resulting data points were then expressed as percentages of the amount of gene 60 synthesized prior to rifampin addition. Logarithmic plots of the resulting data points were made with Microsoft Excel. Exponential regression was used to derive the best-fit curve for the collection of data points over which exponential decay occurred. These curves were y = 146 × e^0.78x (r² = 0.998) and y = 269 × e^0.76x (r² = 0.985), respectively, for gene 60 (Fig. 2A, upper section) test and gap deletion constructs. For GLZ30 test and gap deletion constructs, these were y = 92 × e^0.67x (r² = 0.992) and y = 244 × e^0.54x (r² = 0.997), respectively.

View larger version (46K): [in this window] [in a new window]   FIG. 2.   Bypassing efficiency with constructs containing entire gene 60 coding sequence. (A) Constructs. Abbreviations: Test, test construct; gap, gap deletion construct. (B) Pulse-chase analysis of two independent clones of each construct. Molecular mass indicators in the center are in kilodaltons. The product of bypassing in the left-hand section is 18 kDa. The product of bypassing in the right-hand section is 40 kDa. The left-hand lane of each section shows the product of strains carrying the parental plasmid.

(iii) Functional message accumulation determination. The basic procedure outlined under "Bypass efficiency determination" was followed with the following modifications. Overnight VBMAM-AM cultures were diluted 1:20 into 7 ml of the same medium. Following a 2-h incubation at 37°C, the samples were induced with 2 mM IPTG. Samples (0.5 ml) were removed just prior to induction and then at 2, 5, 10, 30, and 60 min after induction and assayed as described above.

Protein purification and sequencing of internal initiation product. An overnight culture containing the GST-gene 60 gap deletion construct was diluted 1:100 in LB with 2 mM IPTG and incubated for 2 h at 37°C with shaking. Cells (1.5 ml) were recovered by centrifugation and lysed under the conditions described above for the pulse-chase experiments. A 15-µl sample was then run on a Tris-glycine-SDS-15% polyacylamide gel. Following staining with Coomassie blue, the gel was electroblotted onto an Immobilon-P polyvinylidine difluoride 0.2-µm-pore-size transblot membrane. The band of interest was cut out, and the N terminus of the protein was sequenced through nine cycles with an Applied Biosystems model 477 protein sequencer.

Northern blot analysis. Overnight cultures were diluted 1:100 in 10 ml of LB, grown for 2 h, and induced with 2 mM IPTG for 30 min. Total RNA was then purified as described previously (9). Thirty-microgram samples of RNA were loaded per lane, resolved by formamide agarose gel electrophoresis, and visualized by ethidium bromide staining (17). The RNA was transferred by capillary transfer to an Amersham Hybond N⁺ nylon membrane, cross-linked by UV irradiation as recommended by the manufacturer, and hybridized with probe prepared from the 3.1-kb ApaI-SalI lacZ and the 0.7-kb DraI -lactamase fragments of GLZ22 with a Stratagene Prime It-II kit. The blots were then visualized and the amount of hybridization was quantified with a Molecular Dynamics PhosphorImager. The amount of hybridization to -lactamase was used to normalize the amount of lacZ mRNA in each lane.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Amino-terminal fusions. Previously, it has been shown (23) that a small addition of 4 amino acids to the amino terminus of the nascent peptide of gene 60 does not alter bypassing efficiency. We confirmed and extended this finding by adding 1 to 7 amino acids to the amino terminus without observing a significant effect (data not shown). To test if larger additions would alter the nascent peptide signal, GST, gene 10, and protein A-CAT coding regions were added separately to the 5' end of gene 60-lacZ (to produce pSK31, pPG33, and pPA31, respectively) (Table 1). BX3*, which carries 4 codons at the amino terminus, serves as a control in these experiments. The efficiency estimates of 14, 12, 23, and 25% (Table 1) indicate that additions of GST and gene 10 reduce bypassing efficiency twofold. This may be the result of either a reduction in the effectiveness of the nascent peptide signal or simply differences in the levels of stability or functions of mRNAs or protein products of gap deletion constructs. (Note that -Gal activities for pSK31, pPG33, and BX3* test constructs are comparable [4,395, 5,005, and 5,454 U, respectively] but that the values for the gap deletion controls differ [32,000, 41,440, and 23,709 U] [Table 1].)

A new estimate of bypassing efficiency. Test and gap deletion gene 60 constructs were made to measure bypassing efficiency in the absence of any fusion (Fig. 2A, upper section). In these constructs, 45 nt of the 5' wild-type sequence were included so that translation begins from the natural initiation site of gene 60. GST-gene 60 fusions were made to reevaluate whether additions to the amino terminus alter the effectiveness of the nascent peptide signal (Fig. 2A, lower section). The constructs were analyzed by [³⁵S]Met pulse-chase analysis (Fig. 2B), which assays protein synthesis within a 30-s window (see Materials and Methods). Three independent clones per construct were analyzed on multiple separate occasions. Shown here (Fig. 2B) are two representative clones for each construct alongside strains carrying the parental plasmids as controls.

Internal initiation. A secondary product of ca. 16 kDa is apparent in both gene 60 and GST-gene 60 gap deletion lanes (Fig. 2B). The same product is also clearly visible in all test lanes after a longer exposure. To learn its identity, the gap deletion GST-gene 60 construct was overexpressed, the 16-kDa product was purified, and the N terminus was sequenced by Edman degradation. The results (Fig. 3) show that this product results either from internal initiation at Met12 of gene 60 or from proteolysis of the N-terminal region. Previous studies have shown that similarly sized N-terminal deletions of gene 60 reduce bypassing efficiency 10-fold (23). The test 16-kDa product was 5% of the 16-kDa gap deletion product. This finding is consistent with a 10-fold reduction in bypassing efficiency and suggests that this product results from internal initiation. The region around Met12 lacks a strong SD sequence or downstream box, yet the 16-kDa gap deletion band is 30% of the 18-kDa main initiation band, indicating that ribosome loading is fairly efficient from this site. This result implies that internal initiation may distort efficiency estimates in systems where the test-to-gap deletion control ratio is determined by comparing the enzymatic activities of downstream reporter genes.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Amino-terminal sequencing of the gel-purified internal initiation product. The amount of product in each cycle is in picomoles. The derived sequence is boxed.

Distance between landing site and junction with lacZ. While being less than those from previous reports, the above-mentioned estimates are still greater than those obtained with the lacZ fusions described in the first section. One possible variable that may account for these differences is the amount of gene 60 coding information downstream of the landing site. Almost all of the fusions tested above, except for BX3* and the gene 60-phoA fusions, carry 7 codons downstream of the landing site instead of the 2 or 24 codons previously reported (Table 1) (23). To address the issue of whether bypassing efficiency is independent of the distance between the landing site and the junction with lacZ, we compared three isogenic constructs that had 2, 7, and 24 codons between the landing site and lacZ (pGG34, pGG45, and pGG47, respectively) (Table 1). These had bypassing efficiencies of 26, 21, and 28%, respectively, indicating that there are no strict spacing requirements.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Steady-state mRNA levels of GLZ16 and GLZ22 determined by Northern blot analysis. GLZ16 and GLZ22 are isogenic constructs that differ only in their amounts of gene 60 sequence between the landing site and the junction with lacZ (Table 1). Test, test construct; gap, gap deletion construct. Amounts of lacZ mRNA per lane were normalized to the amount of bla mRNA per lane. The data shown represent results of a single experiment (see the text).

Gene 60 functional mRNA stability. Functional message half-lives of test and gap deletion gene 60 mRNAs (Fig. 2A, top section) were determined by pulse-chase analysis of total protein with [³⁵S]Met following rifampin addition (Fig. 5A). Previous work has shown that a lag in the loss of protein synthesis is often observed following rifampin addition (13). This lag is attributed to an increased translation efficiency of longer-lived transcripts as ribosomes released from faster-decaying transcripts increase the available ribosome pool. Following this lag, protein synthesis rates decay exponentially and represent the actual functional half-life of the mRNA (13). Exponential regression was used to model the exponential decay seen from 1 to 3.5 min for test mRNA (Fig. 5A) and from 2 to 5 min for gap deletion mRNA (Fig. 5A). From these tests, functional message half-lives of 0.89 min for test mRNA and 0.91 min for gap deletion mRNA were calculated. Thus, bypassing estimates from these constructs are not distorted by differences in functional mRNA decay rates.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Functional message stability of gene 60 and gene 60-lacZ mRNA. Plots represent the amounts of protein synthesis determined by pulse-chase analysis of induced clones at different times following rifampin addition. Each data point represents the percentage of product synthesized at the given time point relative to the amount synthesized prior to rifampin addition. Error bars represent the standard deviation at each point from results of three independent experiments. Exponential regression was used to generate the curves for each set of data points (given in Materials and Methods). (A) Test (open squares) and gap deletion (filled diamonds) gene 60 (Fig. 2A, upper section) functional mRNA decay. Half lives are 0.89 and 0.91 min, respectively. (B) Test (open squares) and gap deletion (filled diamonds) GLZ30 (Table 1) functional mRNA decay. Half lives are 1.0 and 1.3 min, respectively.

Levels of transcription and accumulation of gap deletion lacZ mRNA. All studies previous to this one have relied on gene 60-lacZ fusions driven by the tac promoter to derive bypassing efficiency estimates. It is possible that high levels of expression titrate a component of the translational machinery that either stimulates or represses bypassing. The constructs discussed above (GLZ16 and pGG34) suggest that efficiency increases with expression levels. To test this suggestion further, we made two sets of constructs that were isogenic except for the promoters. These are GLZ22, GLZ30, pGG36, and pGG34 (Table 1). Bypassing efficiencies for the first two (driven by either the consensus or the tac promoter) are 10 and 32%, respectively. Bypassing efficiencies for the last two (driven by either the lacUV5 or tac promoter) are 18 and 26%, respectively.

View larger version (19K): [in this window] [in a new window]   FIG. 6.   Accumulation of functional mRNA following IPTG induction. Amounts of protein from test mRNA (filled diamonds) and gap deletion mRNA (open squares) were determined by pulse-chase analysis at different time points after IPTG induction and analyzed as percentages of the total cellular protein synthesized. The standard deviation from results of three independent experiments was used to estimate the error for each data point. These data are plotted relative to the scale on the left. Bypassing efficiency (filled triangles) is plotted relative to the scale on the right. (A) GLZ30 (Table 1). Note that this plot does not include the aggregation in the wells mentioned in the text. (B) GST-gene 60. Amounts of product synthesized from test mRNA include both full-length and termination products (Fig. 2A).

Level of ribosome loading. To test if bypassing efficiency is dependent upon translation initiation frequency, we constructed isogenic fusions that differed only in their degrees of Watson and Crick base-pairing potential to the anti-SD region of 16S rRNA (_CGGAAGCU^UCCUCCAC, GLZ32; _AGGAAGCU^UCCUCCAC, GLZ22; _UUGAGGAA^UCCUCCAC, GLZ34; and _AGGAGGUG^UCCUCCAC, GLZ49; where bases with pairing potential appear in boldface type and anti-SD region sequences appear above SD region sequences) (Table 1). In addition, we made a set of constructs that replaced the synthetic SD region with the same 45 nt of the upstream wild-type sequence used for the gene 60 construct shown in Fig. 2A (_AUGAGGUG^UCCUCCAC, GLZ140).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We have shown here that the nascent peptide signal does not require a particular spacing from the amino terminus of the protein to function. Although bypassing efficiency determined from GST-gene 60 fusions (50%) is slightly lower than the efficiency determined from gene 60 that lacks an N-terminal fusion (54%), there are a number of possible explanations for this. Because amino-terminal fusions allow quantification of both failed and successful products of bypassing synthesized from the same mRNA, they will be valuable tools for assaying the effects of various mutations on bypassing efficiency in future studies.

The mechanism by which the nascent peptide signal stimulates bypassing is unknown. It may be that it interacts with the ribosome in the peptide exit channel (7). Interestingly, cryoelectron microscopy experiments have hinted that the 50S subunit may contain more than one exit channel (6, 20) raising the possibility that there are alternate exit paths depending on the character of the growing peptide chain. For instance, proteins destined for export may exit by a different channel than that used for a cytoplasmic protein. In light of this possibility, altering the N-terminal sequence of gene 60 might relocate the emerging nascent peptide within the large subunit. The gene 60-phoA fusions targeting the protein for export suggest that if this is true, it has little bearing on the activity of the nascent peptide signal.

The data presented here support a revised bypassing efficiency estimate of around 50% based on the data from gene 60 and GST-gene 60 constructs (Fig. 2B). These constructs are not subject to the variable functional mRNA levels (Fig. 5 and 6) or internal initiation at Met12 (Fig. 3) that appears to distort efficiency estimates made with gene 60-lacZ fusions. The lack of accumulation of functional gene 60-lacZ test mRNA (expressed from the tac promoter) (Fig. 6A) indicates that the gap may disrupt transcription-translational coupling. This disruption may, in turn, lead to either premature transcription termination or to riboendonuclease cleavage downstream of ribosomes translating lacZ. In either case, ribosomes translating these truncated messages (after bypassing the coding gap successfully) would produce a truncated product not detected by -Gal assays or included in the quantification of protein by pulse-chase analysis. In fact, IPTG-inducible products smaller than the full-length -Gal fusion are present in higher concentrations in clones expressing gene 60-lacZ test constructs than in control constructs (data not shown).

There may be evidence for yet another difference between test and gap deletion constructs that we have not addressed directly. Both gene 60 gap deletion and gene 60-lacZ gap deletion clones have longer lag phases before exponential decay than their test counterparts (Fig. 5). The length of the lag phase has been correlated with translation efficiency (13). This correlation may be an indication of differential ribosome loading rates between test and gap deletion constructs. Alternatively, this might reflect the degree of ribosome protection of downstream message.

The observation that bypassing is significantly less than 100% has two interpretations. The first is that release factors have access to the UAG stop codon just 3' of the takeoff site and that efficiency is determined by the competition between bypassing and termination. Since 1990 (23), any model dealing with bypassing has had to postulate that the UAG codon was inaccessible due to a kinetic or physical barrier erected by the recoding signals. Mechanistically, bypass signals could compete either by interfering with stop codon recognition or by enhancing the dissociation between the peptidyl tRNA and the message. The second interpretation is that all ribosomes initiate bypassing but that only a portion of transiting ribosomes resume translation in the correct reading frame. The termination product (Fig. 2B, right gel) may reflect either a high rate of ribosome dissociation from the message or reinitiation in a different reading frame followed by termination. Experiments to distinguish between these two possibilities are under way.

The results addressing the strength of the ribosome-binding site are intriguing. The increase in bypassing efficiency of 60% upon alteration of a single base pair in the SD sequence suggests that the number of ribosomes upstream of the takeoff site influences the efficiency of bypassing. One possible interpretation is that an upstream ribosome enhances the dissociation of a downstream ribosome complex poised at the takeoff site. Direct ribosome-ribosome contact or application of a force on the mRNA in the 5' direction might be sufficient to stimulate bypassing. There are several alternative explanations. It may be that increased ribosome loading stabilizes test functional mRNA more than gap deletion mRNA by reducing the effects of uncoupling translation from transcription, as postulated above. A second alternative is that strong ribosome initiation at the main initiation site reduces internal initiation at Met12 (Fig. 3), increasing the percentage of bypassing-competent ribosomes. A final alternative requires two assumptions: first, the mRNA can slip in either direction through the ribosome after initiation of bypassing and, second, the slipping back through the ribosome results in unproductive bypassing events. Given these suppositions, upstream ribosomes may limit message movement through the bypassing ribosome to a forward direction and thereby increase the frequency of successful events.

Defining the range of gene 60 bypassing confirms that it is one of the most efficient of all programmed recoding events (which also include programmed frameshifting and stop codon redefinition [7]). Why it occurs at all is still a mystery. T2 and T6 phages have a gene fusion between gene 39 (an upstream topoisomerase subunit gene) and gene 60. While a portion of the coding sequence upstream of the gap remains, the amino terminus (including the critical region) as well as the coding gap are missing (12). This suggests that bypassing is dispensable for phage development. However, it seems that T4 topisomerase II activity is important for high-efficiency replication, as T4 amber mutants defective for gene 60 display cold-sensitive and DNA delay phenotypes (15). There remains the intriguing possibility that bypassing is regulated during T4 infection as a means of enhancing replication; so far, all measurements of bypass efficiency have been done only in uninfected E. coli cells.