d. Mol. Biol. (1990) 213, 811-818

Scanning Model for Translational Reinitiation in Eubacteria Malti R. Adhin and Jan van D u i n l Department of Biochemistry Leiden University Gorlaeus Laboratoria P.O. Box 9502 2300 R A Leiden The Netherlands

(Received 15 August 1989; accepted 19 February 1990) Premature termination of translation in eubacteria, like Escherichia coli, often leads to reinitiation at nearby start codons. Restarts also occur in response to termination at the end of natural coding regions, where they serve to enforce translational coupling between adjacent cistrons. Here, we present a model in which the terminated but not released ribosome reaches neighboring initiation codons by lateral diffusion along the mRNA. The model is based on the finding that introduction of an additional start codon between the termination and the reinitiation site consistently obstructs ribosomes to reach the authentic restart site. Instead, the ribosome now begins protein synthesis at this newly introduced AUG codon. This ribosomal scanning-like movement is bidirectional, has a radius of action of more than 40 nucleotides in the model system used, and activates the first encountered restart site. The ribosomal reach in the upstream direction is less than in the downstream one, probably due to dislodging by elongating ribosomes. The proposed model has parallels with the scanning mechanism postulated for eukaryotic translational initiation and reinitiation.

1. Introduction The initial studies on the mechanism of translational initiation in eukaryotes have revealed substantial differences with eubacteria. For example, eubacteria synthesize proteins from polycistronic messengers, whereas only monocistronic mRNA was found in eukaryotes. In addition, bacteria initiate protein synthesis on AUG, GUG, UUG and AUU, while only AUG seemed to be functional in the eukaryote. A more fundamental difference between the two kingdoms is the capacity of eubacteriM ribosomes to initiate at internal sites, whereas the eukaryotic counterparts were believed to gain access to mRNA only by binding to the (capped) 5' terminus. According to the scanning hypothesis, as formulated by Kozak (1983, 1986a), the eukaryotic ribosomes subsequently migrate along the mRNA until they encounter an AUG codon, whereupon translational initiation occurs, provided that the AUG is embedded in the appropriate context. As time went by, the catalog of eukaryotic messenger sequences grew and exceptions to most t Author for correspondence. 0022-2836/90]120811-08 $03.00]0

rules emerged. The discovery of eukaryotic genes with polycistronic features (Dixon & Hohn, 1984; Hull et al., 1986) broke the monocistronic rule, while the exclusive use of AUG was disproved by the finding that non-AUGs can function in artificial constructs and in higher eukaryotes (Zitomer et al., 1984; Gupta & Patwardhan, 1988). In addition, internal start codons can be reached by a reinitiation mechanism (Kozak, 1984; Liu et al., 1984), originally thought to be the exclusive trait of eubacterial ribosomes. Finally, recent experiments indicate that internal initiation in eukaryotes is a serious possibility (Pelletier & Sonenberg, 1988; Jang et al., 1988; Curran & Kolakofsky, 1989). The mechanism proposed for translational initiation at these internal starts does not, however, violate the basic idea of scanning, but only questions obligatory entry at the 5' physical end of the messenger. These recent observations suggest that important characteristics of start site selection are conserved between the kingdoms. One remaining difference is the capability of the eukaryotic ribosome to arrive at start sites by scanning the message for hundreds of nucleotides, whereas Escherichia coli ribosomes are assumed to bind directly to the initiation region. Here, we present a series of experiments whose 811 ~) 1990 AcademicPress Limited

812

M. R. Adhin and J. van Duin

outcome can best be explained by the assumption t h a t E. coli ribosomes also can reach start sites by migration along the m R N A chain in a scanning-like movement. We have studied reinitiation at the lysis (L) genes of the R N A bacteriophages MS2 and fr. The ribosomal binding sites of the L cistrons are not functional in de novo initiation (Schmidt et al., 1987; Adhin & van Duin, 1989) but their use depends on translation termination at the proximal coat gene (Berkhout et al., 1987; Adhin & van Duin, 1989). I t has been suggested (Sarabhai & Brenner, 1967) t h a t the terminated ribosome reaches the restart site by random m o v e m e n t along the message. To test this hypothesis, we have inserted additional initiation codons between stop and start sites. We show here t h a t these additional start sites function as a barrier to prevent ribosomes reaching the original start. I f translation termination occurs upstream from two competent start sites, the 5' start is used, whereas the 3' start is selected when termination occurs downstream from both start sites. The d a t a are compatible with a model in which translation termination in eubacteria is followed by a bidirectional movement of the ribosome, which m a y result in reinitiation.

2. Materials and Methods (a) Escherichia coli strains Single-stranded DNA for site-directed mutagenesis was isolated from phage M13 derivatives propagated in K12 strain BW313 (dut, ung, thi, relA, spoT1/F'lysA; Kunkel, 1985). Mutants were grown in and isolated from K12 strain JM101 (/ac, pro, supE, thi/F'traD36, proAB, la~Iq, lacZ AM15; Messing & Vieira, 1982). Thermoinducible gene expression was investigated in Kl2 strain M5219 (M72 trpAam, lacZara, Sma/~dbio252, cIs57AH1; Remaut et ed., 1981). (b) Site-directed mutagenesis Oligonucleotide-directed mutagenesis was performed on appropriate M13 subclones as described by Berkhout et al. (1987) and modified by the procedure of Kunkel (1985). The M13 sequencing protocol of Messing & Vieira (1982) was used to detect mutations. The mutated fragments were then placed in their desired context behind the PL promoter. (c) Plasmid descriptions Enzymes used in the construction of the plasmids were purchased from Pharmacia/LKB. All fr plasmids described in this paper contain the fr information downstream from the PL promoter in plasmid pPLa2411 (Adhin et at., 1989). The plasmids denoted as pFRl.n (n=0,1,2 etc.) contain the fr eDNA from nueleotide 9 to 2191 followed by an oligo(dG) region and a PstI site. pFRI.0 contains the wild-type sequence and the changes introduced in pFRI.1 through p F R l . l l are shown in Fig. 2 (below). In pFR1.9, pFRI.10 and p F R I . l l the HhaI site (1565) was opened, subsequently treated with phage T4 DNA polymerase and religated, thus creating a deletion of 2 nueleotides. Nucleotide numbers are taken from Adhin et el. (unpublished results).

The MS2 plasmids described in this study have the phage cDNA inserted in the polylinker of pPLe236 (Remaut et al., 1981). All clones contain the mutation C1657 to A in their phage MS2 DNA. Nueleotide numbers are taken from Fiers et al. (1976). Clone pScl.0 carries the wild-type MS2 EcoRI-BamHI fragment (103-2057) in the corresponding sites of the linker of pPLc236, pScl.l contains 2 substitutions at positions 1690 and 1692 as indicated in Fig. 5 (below). Plasmid pSe2.1 is identical to the corresponding pSel.1 clone except for the deletion of the EcoRI fragment (103-1628). Plasmid pSc4.1 carries the MS2 sequence 1681-2057 preceded by the EcoRI linker sequence GGAATTCC. It contains 2 further substitutions at positions 1690 and 1692 as shown in Fig. 5 (below). The pSc5.n plamids all contain the MS2 eDNA sequence 103-2057. Plasmids pSc5.0 and pSe5.1 have a 1-nucleotide insertion at position 1563 in the coat gene causing premature termination of translation at nucleotide 1652. pSc5.2 and pSc5.3 have 2 nucleotides inserted in the coat gene at positions 1605 and 1608, leading to premature termination of translation at nucleotide 1672. These insertions are described in detail by Berkhout et al. (1987). Further substitutions in the pSc5.n clones are shown in Fig. 5 (below). (d) Detection of lysis gene expression Cell lysis was monitored by measurement of the A650 of cell cultures after induction at 42°C. Production of the coat and lysis proteins was detected on Western blots. Samples were taken 20 min after induction of the PL promoter at 42°C. The solubilized cell extracts were electrophoresed on a 12"5% SDS/PAGE gel (Sch~gger & yon Jagow, 1987) and the proteins were subsequently transferred to nitrocellulose. All L proteins were visualized with an antiserum raised against a synthetic peptide corresponding to the 36 C-terminal amino acids of the MS2 lysis protein. The fr and MS2 coat proteins were detected by an antiserum raised against MS2 coat protein. Immunodetection was performed as described (Towbin et al., 1979).

3. Results (a) Lysis gene expression in bacteriophage fr can be triggered by nearby upstream or downstream translation termination Induction of the wild-type fr cDNA fragment as present in p F R 1 . 0 (Fig. l) leads to the synthesis of coat and lysis proteins as visualized in the immunoblot of Figure 4 (below), where W indicates wild-type-sized lysis protein. The lysis (L) gene of bacteriophage fr begins with the unusual initiation codon UUG and its expression is translationally coupled to the overlapping coat gene. The presence of the unusual start codon restricts de novo initiation at the L start site and expression is triggered b y translation termination at the stop codon of the coat gene (Adhin & van Duin, 1989). This termination dependence is illustrated b y clone p F R 1 . 6 (Fig. 1), in which we have replaced the termination codon of the coat gene by a sense codon causing ribosomes to proceed via eight more triplets to the n e x t in-frame stop at position 1750 (Fig. 2). Clone pFR1.6 fails to lyse (Fig. 3) and synthesis of the L

A Eubacterial Scanning Model

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protein goes down to a virtually undetectable level (Fig. 4). As a possible mechanism for termination-dependent activation of the L gene we envisage t h a t the terminated but not released ribosome reaches the UUG start by sliding along the mRNA, whereupon reinitiation occurs. In the wild-type, this implies a

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ahead of the fr lysis gene Our perception is now t h a t after termination ribosomes diffuse along the R N A until t h e y either spontaneously dissociate or encounter a suitable initiation codon and resume protein synthesis. I f t h i s view is correct, then ribosomes can be prevented from reaching their natural restart site by placing an e x t r a start signal on their course. Accordingly, we introduced an AUG codon 12 nucleotides upstream from and in phase with the L

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net displacement in the 5' direction over 34 nucleotides. As this m o v e m e n t is essentially conceived as a r a n d o m walk of the terminated ribosome along the I~NA it should, to a first approximation, not exhibit directional preference. To test this we constructed clone pFR1.9, in which the coat gene contains a two-nucleotide deletion causing ribosomes to terminate 38 nucleotides upstream from the L start at position 1653 (Figs 1 and 2). Growth curves and immunoblots reveal t h a t lysis expression is indeed activated by termination a t this upstream site, consistent with the presumed reinitiation mechanism (Figs 3 and 4).

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Figure 2. Sequences in the coat-lysis gene overlap region of phage fr in several clones. Start codons that are functional in the respective clones are in bold italics. Stop codons that terminate coat gene translation are underlined and in bold face. E stands for extended lysis protein, W for wild-type-sized and a dash ( - ) for undeteetable amounts of lysis protein.

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(c) Testing the scanning model With two functional restart sites (AUG1679 and UUG1691 ) at our disposal we can now test the assumption that ribosomes "scan" the mRNA until they reach the first start site. Therefore, we constructed clone p F R l . l l , in which both the AUG1679 and the natural UUG1691 are present and coat gene translation terminates upstream from both start sites (Fig. 1). Our model predicts that reinitiation will occur at the first start, AUG16~9. Indeed, Western blot analysis (Fig. 4) shows that only the AUG1679-directed extended lysis protein is present. Apparently, AUG,6~9 prevents ribosomes from reaching the authentic start site (compare pFR1.9 and pFR1.11 ). For the complementary experiment, we constructed pFR1.8, which is identical to the pFR1.11 as used above, except that coat translation now terminates beyond both start sites (Fig. 1). In this clone, UUG1691 is the first start encountered and indeed only UUG169,-directed lysis protein is synthesized (Fig. 4). Ribosomes thus select the first start site on their course, irrespective of whether this site lies upstream or downstream from the termination codon. To generalize our findings we repeated the experiments for another set of genes in which expression is translationally coupled. The coat-lysis genes of RNA bacteriophage MS2 are a suitable pair. The relevant nucleotide sequence in the boundary region is shown in Figure 5 (top line). Although MS2 has the same gene organization as fr, it is sufficiently different to provide additional support for our model. For instance, its lysis gene starts with a regular AUG codon and direct ribosomal access is prohibited by an RNA secondary structure that sequesters the ribosomal binding site (Schmidt et al., 1987). As in fr, expression is triggered by translation termination at the stop codon of the coat gene located 47 nucleotides inside the L gene. Inactiva-

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Figure 4. Western blot analysis of the indicated pFl% clones, showing the size and amount of the synthesized lysis and coat proteins. E stands for extended lysis protein and W for wild-type. C indicates the coat protein. The upper strong band is a background response.

gene by a single C,679 to A substitution. Expression originating from this artificial start will yield a lysis protein that is four amino acids longer. Restart activity of AUG~6v9 was first monitored in clones where the natural UUG,69, was converted into a non-functional UCG codon (indicated by a cross in Fig. 1). Translation termination at position 1653 (pFRl.10) indeed triggers lysis expression from this start, as witnessed by the growth curve (Fig. 3) and the appearance of an extended (E) lysis protein on the Western blot (Fig. 4). AUG,6~9 can also be activated by translation termination at the authentic coat gene stop codon (pFR1.5), albeit with low efficiency (Figs 3 and 4). It is clear that the introduced AUG1679 constitutes a functional start for ribosomes terminating at either side. The activity of AUG,6~9 is still dependent on nearby termination. This is verified by the phenotype of clone pFR1.7, where termination is displaced to position 1750, resulting in completely abolished synthesis of lysis protein (Figs 3 and 4). Activation of both the natural UUG169, and the artificial AUG16v9 is thus

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A Eubacterial Scanning Model

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position 1672 (pSc5.3), AUG16v8 is expected to be used as the restart. This is confirmed by the immunoblot analysis of Figure 6. These findings complement the results obtained in phage ft. 4. Discussion

(a) The eubacterial scanning model

C W S

Figure 6. Immunoblot analysis of the indicated pSc clones, showing the size and amount of the synthesized lysis proteins. S stands for short lysis protein and W for wild-type. The blot is developed with a combination of sera raised against lysis and coat proteins and the variously sized coat-like proteins are marked C.

tion of the stop codon (pMS2H; Fig. 5) inhibits L protein production (Berkhout et al., 1987). On the other hand, premature termination at either 6 or 26 nucleotides upstream from the L start elicits expression (pSc5.2 and pSc5.0; Figs 5 and 6). In fr, the competing start site was placed ahead of the authentic one, while in MS2 we have realized the other possibility by introducing an additional start codon 12 nucleotides downstream from the natural AUG1678. This AUG169o is in the reading frame of the L gene and is preceded by a sequence that can serve as a Shine-Dalgarno (SD) signal (Fig. 5). The potential activity of the artificial start was determined in a construct in which all upstream sequences including the natural lysis start codon had been deleted (pSc4. I; Fig. 5). The new start was found to function, though less efficiently than the natural one. In Figure 6 we show that the resulting shortened lysis protein, designated S, can be distinguished from the wild-type protein marked W. As the next step we constructed a series of clones carrying both start sites. To verify that in these clones L expression is still controlled by coat gene translation we created pSc2.1 in which the start region of the coat gene is absent. Indeed, pSc2.1 synthesizes neither the wild-type nor the truncated lysis protein (Fig. 6), confirming that our assay system meets the demands. In pScl.1, both start sites AUG1678 and AUG169o are present and coat gene translation terminates at its natural stop position 1725. Immunoblots indeed show that in this clone the shorter lysis protein is synthesized (Fig. 6). Some wild-type L protein is also present. Presumably, the introduced AUG169o functions inefficiently as a barrier, resulting in some leakage of ribosomes to the next start. If we arrange termination to occur upstream from both start sites either at position 1652 (pSc5.1) or

In this paper we have analyzed the mechanism of reinitiation at two sets of genes that are transtationally coupled. Extra restart sites were introduced and termination was arranged to occur at different positions in the gene boundary to deduce the relation between stop and restart site. We find that if termination occurs downstream from two competent start sites, the 3' start site is selected, whereas the 5' start is favored when ribosomes terminate upstream from both sites. These results are compatible with a model in which terminated ribosomes remain associated with the message and scan the mRNA in both directions until they either spontaneously dissociate or reinitiate in response to an encounter with a functional start. In the system studied here, dissociation is the more probable event, as the ratio in which coat and lysis proteins are synthesized in wild-type clones is about 20 to 1 (Beremand & Blumenthal, 1979; our unpublished results). Sarabhai & Brenner (1967) were the first to describe translational reinitiation as a non-dissociative event. They discovered the phenomenon in the r l I B gene of phage T4 and suggested that ribosomes drift from the termination to the restart site by "phaseless wandering". We use here the term "scanning" to visualize the analogy we believe to exist with start site selection by the eukaryotic ribosome. Since the pioneering study of Sarabhai and Brenner many reports on the subject have appeared, most of which implicitly or explicitly consider movement of the non-synthesizing ribosome over the RNA lattice (Schiimperli et al., 1982; Ivey-Hoyle & Steege, 1985; Das & Yanofsky, 1984; Baughman & Nomura, 1983; Napoli et al., 1981; Schoner et al., 1984). We and others (Sprengel et al., 1985) have found that restart frequencies dramatically decrease when the distance to the site of termination increases and this result is predicted by the random-walk model. Our studies show for the first time that the restart reach in the upstream direction can be as large as 46 nucleotides. Since movement is here against the direction of translation this implies that scanning must be fast compared to elongation. Still, comparison of the yields in clones pFR1.9 and pRF1.0 indicates that a search in the 5' direction is less efficient than one to the 3' side. Probably, a fraction of upstream scanning ribosomes is dislodged by their elongating counterparts. The prolonged association of the terminated ribosome with the mRNA as proposed by our model need not be surprising. Firstly, Martin & Webster (1975) isolated a termination intermediate

816

M. R. Adhin and J. van Duin

consisting of the 30 S ribosomal subunit attached to mRNA. Secondly, the release of terminated ribosomes from the message is an energy-consuming process (Ryoji et al., 1981, 1985). Finally, polynucleotides like poly(U) and poly(A) and also copolymers have an affinity for ribosomes, irrespective of the presence of an SD sequence (e.g., see Calogero et al., 1988). Schiimperli et al. (1982) have proposed that restarts may result from a local increase in the concentration of free ribosomes around the termination site. In view of the present results we consider this possibility less likely, since such a model predicts a gradient of activity around the termination site rather than titration of all ribosomes at the start site that comes first on a linear scale. For the system used in this paper there are two more observations that argue against mechanisms that involve ribosomes from the pool. Berkhout et al. (1985) displaced the coat gene terminator of MS2 RNA some 200 nucleotides to the 3' side. At the same time the hairpin structure that shields the start of the L gene was broken up by a deletion exposing the start signals. Nevertheless, in such constructs the L gene is not expressed unless the flow of ribosomes is interrupted before moving across the start of the L gene. This is not a peculiarity of the L gene. The replicase gene starting somewhat further downstream is also silenced when overrun by elongating ribosomes. Apparently, the flow of ribosomes over the coat gene is so dense that it prevents de novo initiation from the pool. Findings similar to these have been reported by Das & Yanofsky (1984). For phage fr there is an additional reason to disregard the two-ribosome model. Its L cistron begins with UUG and, at least in the fr context, this codon does not measurably function in primary initiation. For instance, fr subclones containing the L cistron but lacking upstream translation from the coat gene do not express the L protein unless UUG is replaced by GUG or AUG (Adhin & van Duin, 1989). Finally, it should be mentioned that in many of our constructs stop and restart sites are simply too close to accommodate two ribosomes. (b) E~ciency of reinitiation Of the possible parameters that influence the efficiency of translational reinitiation two will be discussed here. One is the presence of a ShineDalgarno sequence. During this work we have noticed that not every AUG created in the vicinity of the termination codon functions as a restart signal. Only those preceded by an SD-like sequence gave measurable restart activity. We have recently shown using another system that reinitiation works about ten times better in the presence of an SD region (Spanjaard & van Duin, 1989). Another important factor is the distance between stop and restart site. It is not clear why some systems like the r I I B gene in T4 show a sharp drop of activity around the termination codon, whereas

others, including the one studied by us, do not. One reason could be that in the short-range systems lateral diffusion is impaired by secondary structure. Apart from one or two exotic exceptions (Huang et al., 1988; Gold, 1988) translating ribosomes unfold RNA hairpin structures on their course (Mayford & Weisblum, 1985; Yanofsky, 1981). The energy required for this process is probably provided by GTP hydrolysis that drives protein synthesis. As scanning is a random process not considered to consume energy we expect it to be restricted by base-paired regions. Evidence to support this statement can be derived from results reported by Schmidt et al. (1987). In Figure 7, we present the RNA secondary structure in the start region of the L gene of MS2 RNA as determined by chemical and enzymatic modification, phylogenetic sequence comparison and phenotypes of constructed mutants (Schmidt et al., 1987). One of the constructed mutants, pMS2.d4, carries two substitutions that stabilize the hairpin and this mutant fails to synthesize L protein in response to termination at the natural stop signal of the coat gene. Presumably, scanning ribosomes are unable to melt the stabilized stem-loop structure. To verify that the substitutions had not inactivated the start site by primary sequence effects, derivatives of pMS2.d4 were constructed in which termination was arranged to occur in the stem-loop structure itself (positions 1652 or 1672, boxed in Fig. 7). These clones do express the L protein and the results therefore suggest that stem-loop structures indeed impair scanning. (c) Comparison of eubacterial and eukaryotic scanning Formally, two types of messenger scanning have been described in the eukaryotic system. One starts from the favored ribosome binding site in eukaryotic messengers, the 5' physical end, and proceeds in the (only possible) 3' direction until a start site is reached. The other kind occurs after termination. Upon hitting a stop codon, ribosomes release their protein and apparently stick to the message and resume scanning until they either meet a restart site (Liu et al., 1984; Kozak, 1984) or dissociate. There are no data to indicate that the two kinds of scanning are basically different. It is therefore surprising that reinitiation at upstream positions was reported to take place in eukaryotes {Peabody & Berg, 1986; Peabody et al., 1986), implying that the movement of eukaryotic ribosomes with respect to the message is random rather than unidirectional. If so, this would be a remarkable parallel between eukaryotic and eubacterial translation. A quantitative difference between bacterial and eukaryotic scanning is the distance that can be screened. In eukaryotes this is far greater, both in cap-directed (Kozak, 1983) and in terminationdependent starts (Liu et al., 1984). This difference is probably due to the presence of initiation factors that melt hairpin structures in an energy-requiring

817

A Eubacterial Scanning Model

efficiency with which sequence-specific proteins (lac repressor, E c o R I endonuclease) locate their recognition sites (Berg et al., 1981; T e r r y el al., 1985). I t is conceivable t h a t internal initiation of eukaryotic ribosomes proceeds in a similar fashion (Pelletier & Sonenberg, 1988). The results presented here reveal an unexpected parallel between eubacterial and eukaryotic ribosomes in their search for initiation sites and support the notion t h a t fundamental mechanisms are conserved through all kingdoms.

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We thank J. Alblas for assistance, V. Berzin for the partial fr eDNA clone, and W. Fiefs and E. Remaut for the supply of MS2 eDNA and the expression vector pPLc236.

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Figure 7. RNA secondary structure in the lysis gene initiation region of phage MS2. The base substitutions in mutant pMS2.d4 are indicated with arrows. The lysis start and the coat gene termination codon are indicated by a continuous line. Out-of-phase termination codons used in control constructs are boxed.

process (Kozak, 1986b; Sonenberg, 1988). I t is thus possible t h a t the eukaryotic ribosome proper is not able to travel further t h a n its bacterial homolog. I f scanning is a general p r o p e r t y of the eubacterial 30 S subunit, then it is likely t h a t de novo initiation in eubacteria is not necessarily mediated by direct binding of the ribosome to the s t a r t site. Instead, we can envisage a situation where productive initiation occurs even when ribosomes contact the R N A in the vicinity of the start site from where they m a y drift to their target. There is evidence for this scenario. Wulff et al. (1984) and Cone & Steege (1985) have reported t h a t neighboring out-of-phase start codons inhibit translation by competing with the authentic start. The in vitro equivalent for this observation is t h a t the position of the ribosome in a translational initiation region can be changed to neighboring codons b y replacing the initiator t R N A by t R N A cognate for those other codons (Hartz et al., 1988). These findings illustrate the capacity of a ribosome, in this case tethered b y an SD sequence, to move relative to the message. Such an e n t r y of the ribosome bears resemblance with the linear diffusion mechanism proposed for the high kinetic

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Edited by S. Brenner

Scanning model for translational reinitiation in eubacteria.

Premature termination of translation in eubacteria, like Escherichia coli, often leads to reinitiation at nearby start codons. Restarts also occur in ...
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