The role of the leader sequence coding region in expression and assembly of bacteriorhodopsin.

Bacterio-opsin is made as a precursor in Halobacterium halobium, which has 13 additional residues at the amino terminus. The codons for these residues have been proposed to form a hairpin structure in the mRNA and play a role in ribosome binding; the leader peptide sequence also has been proposed to have a role in membrane insertion of bacteriorhodopsin (BR). We have made mutations in the bop gene region coding for the leader sequence and expressed the mutant genes in an H. halobium mutant lacking wild-type BR. The leader sequence coding region was found to be important for the stability of the mRNA and for its efficient translation. Single base substitutions in this region that did not affect the amino acid sequence caused significant reductions in protein expression. Deletion of the leader region resulted in unstable mRNA and almost no BR production. Introduction of a new ribosome-binding sequence within the coding region of the mature protein restored mRNA stability and some protein expression. Protein made without the leader peptide was properly assembled in the membrane.

Bacterio-opsin is made as a precursor in Halobacterium halobium, which has 13 additional residues at the amino terminus. The codons for these residues have been proposed to form a hairpin structure in the mRNA and play a role in ribosome binding; the leader peptide sequence also has been proposed to have a role in membrane insertion of bacteriorhodopsin (BR). We have made mutations in the bop gene region coding for the leader sequence and expressed the mutant genes in an H. halobium mutant lacking wild-type BR. The leader sequence coding region was found to be important for the stability of the mRNA and for its efficient translation. Single base substitutions in this region that did not affect the amino acid sequence caused significant reductions in protein expression. Deletion of the leader region resulted in unstable mRNA and almost no BR production. Introduction of a new ribosome-binding sequence within the coding region of the mature protein restored mRNA stability and some protein expression. Protein made without the leader peptide was properly assembled in the membrane.
Bacteriorhodopsin (BR) 1 is a light-driven proton pump in the membrane of the archaeon Halobacterium halobium (also known as Halobacterium salinarium). Bacterio-opsin (BO), the apoprotein without the retinal chromophore, is made as a precursor that has 13 additional residues at the amino terminus (1). These residues, as well as one at the carboxyl terminus, are removed after membrane assembly (1,2). The leader sequence does not have the positive amino-terminal and hydrophobic domain characteristic of both prokaryotic and eukaryotic signal sequences and is also too short to span the bilayer. Nevertheless, it has been proposed to have a role in the insertion of BR into the halobacterial membrane (3,4). Mature BR lacking this sequence inserts spontaneously into phospholipid bilayers in vitro (5)(6)(7)(8)(9), and BO synthesized in vitro in a wheat germ system is integrated into dog pancreas microsomes equally well both with and without the leader sequence (10). These processes may, however, be different from what occurs in the H. halo-bium cell. The mRNA transcribed from the bop gene coding for BO, as well as mRNAs for some other H. halobium proteins, starts very close to the initiation codon and has been proposed to form a stem and loop secondary structure containing a sequence complementary to the H. halobium 16S rRNA ( Fig. 1) that could act as a ribosome-binding site within the coding region for the protein (1,11,12). Our experiments have confirmed this role for the 5Ј end of the bop mRNA that codes for the leader sequence, but we have found no requirement for the leader sequence itself in membrane insertion or assembly of BR.

EXPERIMENTAL PROCEDURES
Materials-Lovastatin (also known as mevinolin) was a gift from Dr. A. W. Alberts (Merck Sharp and Dohme). NspI (Nsp7524 I) was obtained from Amersham Corp. Recombinant DNA procedures were carried out as described in Ref. 13 or 14 using enzymes and kits available from various commercial sources except as described below.
Plasmid Constructions-The bop gene from H. halobium strain ET1001 (provided by Dr. R. Bogomolni, UC Santa Cruz) was cloned by probing a library of size-selected BamHI-NotI genomic fragments in pBS/KS(ϩ) (Stratagene, La Jolla, CA) with the oligonucleotide 5Ј-CGC-CCATCCCTTTCACGAG. One of the positive clones was sequenced, and the bop coding region was found to be identical to the previously published sequence from an unidentified strain (1). The BamHI-NotI fragment containing the bop gene was excised from the vector and ligated to the BamHI and HindIII sites of pUC19 (15) using the adapter oligonucleotides 5Ј-GGCCGCGACCAGCGACTGATCTAGA and 5Ј-AGCTTCTAGATCAGTCGCTGGTCGC, which replace the last few codons of the bop gene 3Ј of the NotI site and create XbaI and HindIII sites just 3Ј of the termination codon. The BamHI-XbaI fragment containing the bop gene was cut out of this plasmid (designated pBO6) and ligated between the BamHI and XbaI sites of the shuttle vector pWL102 (16), which contains a gene conferring resistance to the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor lovastatin in H. halobium, as well as a ␤-lactamase gene and an Escherichia coli origin of replication. The resulting plasmid was designated pXU3. An alternative strategy was also used in which the bop gene was excised from pBO6 as an EcoRI-HindIII fragment and inserted between the corresponding sites on pWL102. This results in deletion of the 3.8-kb HindIII fragment of pWL102 derived from pHV2 (16) that had been inserted to allow replication of the plasmid in halobacteria. In our hands this fragment was unnecessary, because all of our pWL102-based constructs inserted into the chromosome and did not replicate as plasmids in H. halobium.
Mutagenesis-Deletion of the leader sequence coding region was accomplished by replacing the NspI-AatII fragment of pXU3 that codes for the leader sequence and the first few amino acids of the mature protein with synthetic linkers. The oligonucleotides 5Ј-CAGGCCCA-GATCACCGGACGT and 5Ј-CCGGTGATCTGGGCCTGCATG were used to make pXU10 (see Fig. 2), and pXU10A was made using 5Ј-AACGTCCAGGTGACCGGACGT and 5Ј-CCGGTCACCTGGACGT-TCATG. Single base substitutions in the leader sequence coding region were made by amplifying the region of the bop gene from base 356 to 878 (numbering according to GenBank entry HHABO, where base 361 is the A of the ATG initiation codon) using the polymerase chain reaction with a mutagenic primer for the coding strand and the primer 5Ј-GCCTTCGAGGTGAACCCG for the complementary strand. The sense strand primers were: 5Ј-GTTGCATGTTGAAGTTATTGCC (G 367 3 A) for pXU13, 5Ј-GTTGCATGTTGGAATTATTGCC (G 369 3 A) for pXU14, and 5Ј-GTTGCATGTTAGAGTTATTGCC (G 366 3 A) for pXU15 (see Fig. 5). Amplifications were done for 30 cycles (1 min at 94°C, 1 min at 55°C, and 2 min at 72°C) using Pfu Polymerase (Stratagene) and pBO6 as a template in a variety of different thermal cyclers. The polymerase chain reaction amplification products were cut with NspI and KpnI, purified by agarose gel electrophoresis, and then ligated to pWL102 together with the BamHI-NspI and KpnI-XbaI fragments of the wild-type bop gene in pXU3. Mutations were confirmed by sequencing the plasmids prior to transformation of H. halobium.
Transformation-H. halobium strains SD9 and SD16 (17), which have a 1-kb ISH1 and a 0.5-kb ISH2 insertion, respectively, in the coding region of the bop gene, were transformed using the plasmids described above. The transformation procedure was essentially as described in Ref. 18. Briefly, 20 ml of log phase cells were spun down and resuspended in 2 ml of spheroplasting solution. 0.2-ml aliquots were added to 20 l of 0.5 M EDTA in spheroplasting solution and gently agitated; this converted the cells to spheroplasts in less than 10 min for the strains we used. After 15 min, 5-10 g of DNA in 20 l was added, followed 5 min later by 240 l of 60% polyethylene glycol 600 in spheroplasting solution; mixing was accomplished by gently tilting the tube back and forth. After 20 min of additional incubation at room temperature, 1 ml of regeneration salt solution was added. Cells were pelleted in a microcentrifuge at 4,000 rpm for 5 min, then resuspended in 1 ml of complex media (19) containing 15% sucrose, and incubated 18 h at 37°C. 100-l samples were spread on plates containing complex media and 15% sucrose without drug and incubated for 5 days at 40°C. The bacteria were then harvested from the plates and spread on plates containing complex media and 16 g/ml lovastatin. Alternatively, the transformed cells were incubated 2-3 days in complex media with sucrose and then spread directly on plates with drug. After about 10 days of incubation at 40°C, colonies were picked and grown in complex media with 16 g/ml lovastatin, and their DNA and proteins were analyzed.
Southern Blots-Chromosomal DNA was isolated from H. halobium by a procedure similar to methods used for bacteria (13,14) involving lysis in 0.25 M Tris, 0.05 M EDTA, pH 8, followed by digestion with RNase A followed by digestion with proteinase K in the presence of 1% SDS, extractions with phenol, phenol/CHCl 3 , CHCl 3 , and ether, and two ethanol precipitations. H. halobium DNAs were digested with either SmaI or BamHI ϩ NotI and run on 0.8% agarose gels in TAE buffer (13). The separated fragments were transferred to nitrocellulose or nylon membranes by Southern blotting (13), and the DNA was fixed to the membranes by microwaving the dried membranes for 2 min on "high". Blots were hybridized first with a probe made from the BamHI-XbaI fragment containing the bop gene (see above) by nick translation or random priming using [␣-32 P]dCTP. Following autoradiography the blots were stripped and then reprobed with the synthetic oligonucleotides that had been used to make the mutation, which were labeled using [␥-32 P]ATP and T4 polynucleotide kinase. The second hybridization was carried out at 50 -60°C, and the washes that followed were in 6 ϫ SSC at 37°C. Under these stringent conditions, the probes did not bind to wild-type bop DNA sequences on control lanes of the blot.
Northern Blots-H. halobium mRNA was isolated by a procedure recommended for Gram-positive bacteria (13). RNAs were separated on 1% agarose gels containing formaldehyde and transferred to nitrocellulose by Northern blotting (13). The blots were probed with the same nick-translated or random-primed bop gene probes used for the Southern blots.
Quantitation of BR-BR makes up a substantial fraction of the membrane protein in the H. halobium strains used in this study when they have a functional copy of the wild-type bop gene; the yield of BR upon purification by standard procedures (19) is dependent on the level of BR expression, because the formation of purple membrane patches requires a high density of BR in the membrane. We therefore quantitated the amounts of BR in the membranes of various mutant strains by looking at the amount of BR relative to other proteins and chromophores in crude membrane preparations. H. halobium strains were grown for 3 days after reaching stationary phase, and equivalent numbers of cells were used to prepare membranes as follows. Cell suspensions (25-50 ml) were centrifuged at 3000 ϫ g for 10 min; the cells were then lysed by resuspending the pellet in 2 ml of H 2 O and 0.1 mg DNase I and sonicating for 1-2 min using a cup horn. Membranes were pelleted by centrifugation for 1 h at 180,000 ϫ g; the membrane pellet was resuspended in 2 ml of distilled H 2 O by sonication, recentrifuged twice, and then resuspended in 0.5-1 ml of H 2 O for spectrophotometric analysis. Spectra were obtained on a Varian Cary 3 at room temperature; the absorbance at 410 nm due to cytochromes in the membranes served as an internal standard to adjust the concentration of membranes used in each sample. Aliquots were also analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and staining with Coomassie Blue or silver. In some cases proteins were transferred to polyvinylidene difluoride membranes by Western blotting and probed with rabbit antibodies to BR, followed by goat anti-rabbit Ig conjugated to HRP and then color development using 4-chloronaphthol and H 2 O 2 . The soluble fraction of the cell lysate was also examined by these methods.
Pulse-Chase Experiments-50-ml cultures of H. halobium were grown to an A 600 of 1.0 under fluorescent lights in complex media and 4 g/ml lovastatin. The cells were spun down at 1,000 ϫ g for 15 min and resuspended in 10 ml of basal salts (medium without peptone; Ref. 19) and 2.5 mg/ml L-alanine in a 15-ml conical tube. The tubes were rotated at 40°C under fluorescent lights for 2 h; then 2 Ci of [ 35 S]methionine (Ͼ800 Ci/mmol) was added. After 2 h of incubation, a 2-ml aliquot was removed (pulse) and unlabeled L-methionine was added to the remaining 8 ml at a final concentration of 5 mg/ml. Incubation was continued and additional 2-ml samples were removed at times ranging from 2 to 48 h after addition of the chase. Each aliquot removed was immediately spun down at 5,000 ϫ g for 5 min; the cells were lysed by addition of 0.5 ml of H 2 O and 0.1 ml 2.5 mg/ml DNase I and sonicated for 1 min at 0°C in a cup horn. Membranes were pelleted by centrifugation at 180,000 ϫ g for 30 min at 4°C. Pellets were resuspended in 50 l of H 2 O; 125 l of acetone (Ϫ20°C) were added, mixed, and centrifuged at 16,000 ϫ g for 30 min at 4°C. The final pellet was resuspended and boiled for 5 min in 10 l of gel loading buffer containing 2% SDS. Samples were electrophoresed on 17.5% acrylamide gels, which were fixed or stained and dried, and then exposed to a PhosphorImager cassette for 6 -15 days. The phosphor screens were scanned in a Molecular Dynamics 400E PhosphorImager and analyzed using Image Quant software.

RESULTS
Transformation of H. halobium Strains-In order to determine the role of the leader sequence in BR expression and membrane assembly, mutant bop genes had to be expressed in a H. halobium strain that did not make wild-type BR. Strains SD16 and SD9 (17) were chosen for this purpose. SD16 has a 520-base pair ISH2 insertion near the 3Ј end of the bop gene, whereas SD9 has a 1,118-base pair ISH1 insertion at the 5Ј end of the bop gene coding region (17). SD16 and SD9 cells were transformed with recombinant plasmids containing the wildtype bop gene (pXU3) or mutant bop genes (pXU10 -15). Transformations resulted in lovastatin-resistant colonies that were either predominantly purple (due to production of BR) or orange-colored, like SD16 and SD9 cells. Growth of the transformed cells at the high levels of drug used for selection (16 g/ml) was very slow, but control cells treated without the plasmid produced few or no colonies on the drug plates and less than 1% of the transformants with pXU3 were spontaneous drug-resistant mutants (orange colonies). In contrast, using 4 g/ml lovastatin resulted in faster growth of transformed cells but high levels of spontaneously resistant cells in transformations without DNA and 50% or more orange colonies with pXU3.
Whereas pWL102 is stably maintained in H. halobium, the presence of a bop gene insert in each case caused the plasmid to integrate into the chromosome at the site of the endogenous bop gene. DNA from the transformants was analyzed by digestion with SmaI, Southern blotting, and probing with a nicktranslated bop gene fragment. SmaI sites are located 450 base pairs upstream and 1.7 kb downstream of the bop gene coding region, so in strains SD16 and SD9 the bop probe hybridizes with 3.5-and 4-kb fragments, respectively. All the transformants with pXU3 and pXU10 -15 that were analyzed had a single band of Ն15 kb hybridizing to the probe in the SmaI digests, indicating that the entire plasmid was integrated into the chromosome at the bop locus.
DNA from the transformed SD16 and SD9 cells was also analyzed by digestion with BamHI and NotI. The BamHI site is located 388 bases upstream of the initiation codon, and the NotI site is located 16 bases upstream of the termination codon, so that the BamHI-NotI fragment containing the bop coding region is about 1.2 kb in the wild-type gene and the recombinant genes on the plasmids but 0.5-1.1 kb larger in the SD16 and SD9 chromosomes (with their ISH insertions). Southern blots consistently showed two bands (1.2 and 1.7 or 2.3 kb) hybridizing with a nick-translated bop gene probe. For the transformants with pXU10 and the other mutant bop genes, the question remained whether recombination between the plasmid and the chromosome had occurred upstream or downstream of the mutation, because the latter would result in regeneration of a wild-type gene in the chromosome. The blots were stripped and reprobed with an oligonucleotide specific for the mutated sequence in order to determine which one of the two genes had the mutation, the one inactivated with the insertion (the larger BamHI-NotI fragment) or the one with that could be transcribed and translated to make the protein (1.2-kb band).
In order to maximize the odds of obtaining transformants with functional mutant genes and inactivated wild-type genes, transformations with mutant bop genes were done using strain SD9. Because the ISH1 insertion is near the location of all the mutations introduced into the bop gene on the plasmids, recombination between the plasmid and the chromosome both upstream of the mutation (in the 5Ј-flanking region) or downstream of the mutation (and of the ISH1 insertion in the chromosome) would result in the mutant gene remaining intact. SD9 cells transformed with the various plasmids containing wild-type and mutant bop genes were analyzed using Southern blots of BamHI ϩ NotI digests that were first probed with a random-primed bop gene and then stripped and reprobed with an oligonucleotide specific for the mutant sequence. In each case the smaller (1.2 kb) band containing the uninterrupted bop gene hybridized to the mutant probe, whereas the larger (2.3 kb) band containing the ISH1 insertion did not. This is shown for two transformants with pXU10A in Fig. 3. Transformations of SD16 with mutant bop genes, on the other hand, frequently resulted in regeneration of a wild-type bop gene.
The Leader Sequence Coding Region Is Required for BR Expression-SD16 and SD9 cells transformed with pXU3 (the latter is referred to as strain XU3 below) produced BR at levels comparable to that of strain S9, the BR-overproducing strain from which SD9 and SD16 had been derived (17). However, SD9 cells transformed with pXU10 (strain XU10), the leader sequence deletion mutant (Fig. 2), did not produce BR at levels detectable by SDS-PAGE using Coomassie Blue or silver staining or by examination of the visible spectrum of membranes from XU10 cells (not shown). Western blots of XU10 membranes showed extremely faint bands that co-migrated with BR, which could not be reproduced photographically.
In order to determine whether this lack of BR expression was due to lack of transcription of the bop gene with the deletion or lack of translation of the mRNA, RNA was isolated from XU10 and XU3 cells and analyzed by Northern blotting (Fig. 4). The bop mRNA in XU10 cells was found to be degraded to low molecular weight fragments that nevertheless still hybridized to the bop probe; the XU3 cells were always found to have only intact bop mRNA with the expected molecular weight as shown in Fig. 4.
Point Mutations in the Proposed Ribosome-binding Site Affect BR Expression-In order to test the theory that the bases coding for the leader peptide at the 5Ј end of the bop mRNA also act as the ribosome-binding site and form a hairpin secondary structure, three point mutations were introduced in this part of the sequence (Fig. 5). In each case a single G in pXU3 was changed to an A by polymerase chain reaction mutagenesis to make pXU13, pXU14, and pXU15; these were used to transform SD9 cells and create strains XU13, XU14, and XU15, respectively, and proper integration of each intact mutant gene in the chromosome checked by Southern blot analysis as described above. In XU13 the mutation results in the third amino acid residue of the leader sequence changing from Glu to Lys; XU14 and XU15 have silent mutations, so any changes in BR expression in these strains cannot be due to altered protein folding, processing or membrane assembly. All three mutations cause mismatches in the proposed base pairing of the mRNA to the H. halobium 16S rRNA (Fig. 5); the mutations in XU13 and XU15 also decrease the stability of the proposed hairpin stem secondary structure.
Colonies and cultures of the three mutant strains appeared less purple than those of XU3 or S9. The amount of BR produced by strains XU13, XU14, and XU15 was compared with that of XU3, which has the same genetic background but no mutation in the bop gene, by looking at the spectra of membranes prepared from similar amounts of cells (Fig. 6). The spectra showed that XU13, XU14, and XU15 cells produced less than half as much BR as XU3. The experiment was repeated three times, and the relative amounts of BR (570 nm absorbance) found in strains XU13, XU14, and XU15 varied slightly but was consistently less than 50% of that in XU3 and distinctly more than in XU10A. The relative amounts of BR in the various strains was also compared by examining the intensities of the BO polypeptide bands when SDS-PAGE gels were run on the membranes (Fig. 7). The intensities of the bands on the SDS-PAGE gel shown in Fig. 7 did not show much difference between XU13 and XU14, and the band for XU10A is barely detectable on the photograph. These results were consistent with the spectra taken on that set of membrane samples, which were not the ones used for Fig. 6. The gels also showed that the BR in all the strains, including that of XU13 with its altered leader peptide sequence, was fully processed to the mature form by removal of the 13 amino-terminal amino acids. The soluble fractions of the cells (not shown) did not contain any BR.
Northern blots showed that the bop mRNA in XU13, XU14, and XU15 was the same size as in XU3 and was not degraded like that of XU10 (Fig. 4). The variations in intensity between the bands were probably due to differences in yield during RNA isolation and were reflected in the intensity of the total RNA staining with ethidium bromide in each lane on the gel used for the Northern blot (not shown).
Introduction of a New Ribosome-binding Site in the Leader Deletion Mutant Restores mRNA Stability and Some BR Expression-In order to test further the hypothesis that the coding region for the leader sequence at the 5Ј end of the mRNA functions as a ribosome-binding site and is important for mRNA stability, we constructed a new mutant, designated XU10A. This mutant had the same deletion of the codons for the leader sequence as XU10 and additional mutations in the region coding for the first four residues of the mature protein that resulted in the 5Ј end of the mRNA again having complementarity to the 16S rRNA and the potential to make a stem and loop secondary structure (see Fig. 2). These were either silent mutations or conservative substitutions (Gln 3 Asn, Ile 3 Val, Ala 3 Val) in order to minimize any effects on protein folding or stability. This amino-terminal region of the protein protrudes on the exterior side of the membrane in H. halobium and is not resolved in the three-dimensional structure of wildtype BR (20).
XU10A cells were pink, in contrast to the orange SD9 and XU10 or the purple XU3 cultures. Northern blots showed no degradation for XU10A mRNA (Fig. 4). The spectrum of XU10A membranes showed a peak at 570 nm characteristic of BR, which appeared as a shoulder on the carotenoid peak similar to that in XU14 membranes (Fig. 6). A faint BR band could also be detected on SDS-PAGE gels (Fig. 7). The relative amounts of polypeptide detected on gels are consistent with the amounts of the chromophore detected in spectra, indicating that most or all of the BO produced had bound retinal to form BR, which produces the characteristic absorption spectrum. No BR could be detected in spectra nor gels run on the soluble fraction of XU10A cells (not shown).
Pulse-Chase Experiments Indicate That There Is No Increased Turnover of BR in XU10A-XU3, XU10A, and XU14 cells were pulse-labeled with [ 35 S]methionine followed by a chase with unlabeled methionine, and the labeled proteins examined by SDS-PAGE and PhosphorImager analysis (Fig. 8). In all three strains no significant turnover of BR in the membranes could be detected over a period of 48 h after dilution of the label. The amount of label incorporated was lowest in XU10A and highest in XU3, reflecting the amounts of BR found in the strains cultured in rich media (see above and Figs. 6 and 7). No labeled BR was ever found in the soluble fractions of XU3, XU14, or XU10A cells. DISCUSSION Transformation of H. halobium SD16 and SD9 with the wild-type bop gene fully restored expression of BR; this was not surprising, because the wild-type gene had been inserted back into the chromosome at the bop locus. Efficient expression of the wild-type bop gene in strain L33 (which has an ISH2 insertion in the bop coding region like SD16) using a similar plasmid has been reported previously by Needleman's group (21). Their plasmid also contained the brp gene upstream of bop and was usually, but not always, found integrated into the chromosome (21). The reason we never found transformants with plasmids containing a bop gene that had not integrated into the chromosome may be a loss of unintegrated plasmids during the prolonged incubation period without drug selection.
The lack of BR production in the leader sequence deletion mutant XU10 was most likely caused by the degradation of the mRNA and perhaps lack of translation of any remaining message due to deletion of the putative ribosome-binding site, which is in the region coding for the leader sequence (see Figs.  1 and 2). This ribosome-binding site is downstream of the initiation codon, unlike those of eubacteria, which are upstream of the initiation codon but similarly complementary to the 3Ј end of the 16S rRNA. Secondary structure and ribosome binding at the 5Ј end of the bop mRNA apparently enhances the efficiency of translation by an unknown mechanism but does not interfere with the binding of complementary tRNAs to these bases when translation is initiated.
Strains XU14 and XU15 have only silent mutations and produce wild-type BR; their bop mRNA was produced in normal amounts and was not found degraded, so the reduced level of BR expression in these strains must be due to decreased translation of bop mRNA. In the case of XU13, the presence of an amino acid substitution that created a major difference in the net charge of the leader peptide did not cause any additional reduction in the amount of BR found compared with XU15. Both XU13 and XU15 have mRNA sequences that are predicted to have reduced binding to the 16S rRNA as well as reduced stability of the mRNA secondary structure. This perturbation of the hairpin secondary structure would not occur in XU14, but the level of BR in this mutant was on average comparable with that of XU13 and XU15. This suggests that the ribosome-binding sequence determines the level of translation and the exact secondary structure of the hairpin may not be critical for translation. The presence of a hairpin may, however, be required to prevent rapid mRNA degradation.
The results obtained with the modified leader sequence deletion mutant XU10A indicate that the presence of a ribosomebinding sequence and/or hairpin loop at the 5Ј end of the bop mRNA in XU10A was sufficient to stabilize the mRNA and facilitate a modest level of translation. The reason that the level of BR expression was significantly less than that in XU3 FIG. 6. Spectra of membranes from H. halobium mutants. The relative absorbance at 568 nm (due to bacteriorhodopsin) gives an indication of the relative amounts of BR produced in each strain. Absorbances at 410 nm due to cytochromes and at 470 -550 nm due to carotenoids are also visible. Spectra of membranes from strains XU3, XU15, XU13, XU14, XU10A, and SD9 (the parent strain) transformed with the vector pWL102 are shown. may be because the new ribosome-binding site and secondary structure are not very close replicas of those in the wild-type bop gene. In fact, the structure of XU10A mRNA is in some ways more similar to that of the hop gene mRNA (see Fig. 1). This gene, coding for the protein halorhodopsin, is expressed at much lower levels than bop (22). Consistent with this interpretation is the observation that bop-hop fusions in which the 5Ј untranslated region (including the promoter) of the bop gene is fused to the hop coding region do not significantly increase the level of hop expression, but fusions that include a portion of the bop coding region (and therefore the bop ribosome-binding site) result in a high level of production of halorhodopsin (23). 2 Similarly, fusion of the bop promoter to the sopI (sensory opsin I) gene does not significantly increase expression of sopI, but fusions that also include the first 13 or 21 codons of the bop gene result in much higher expression levels (24).
The BR produced in XU10A was assembled with the retinal chromophore, and it was all found integrated into the H. halobium membrane. We were unable to extract it from the other membrane components by high salt washes or density gradient centrifugation. Therefore, the leader peptide must not be required for proper folding or membrane insertion. It may, however, facilitate these processes in wild-type cells (i.e. the leader peptide may have a role in the kinetics of protein folding or membrane insertion). In either case, the absence of the leader may result in misfolding or improper targeting of a substantial fraction of the newly synthesized polypeptide. Ribosomes translating the bop gene have been found bound to membranes and to a 7S RNA with a proposed signal recognition particle (SRP)-like function (4); these interactions may involve the leader sequence and enhance the efficiency of membrane insertion. An altered amino-terminal sequence might also result in increased turnover of the BR in the membrane, but this was not observed.
The pulse-chase results suggest that the level of BR expression is determined by the rate of synthesis. Unfortunately, the rate of incorporation of labeled methionine into BR in XU10A cells was too slow to allow pulses shorter than 2 h. Therefore one cannot rule out the possibility that much of the BO polypeptide in this strain is rapidly degraded before it has a chance to be integrated into the membrane, because it is inefficiently translocated or misfolds in the absence of the leader sequence.