Friedrich-Schiller-Universität Jena, Institut für Molekularbiologie, Winzerlaer Straße 101, biolitec AG, Winzerlaer Straße 2a2, Jena D-07745, Germany
Author for correspondence: Sabine Brantl. Tel: +49 3641 657576/78. Fax: +49 3641 657520. e-mail: Sabine.Brantl{at}rz.uni-jena.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() |
---|
Keywords: plasmid copy number control, circular dichroism measurements, Gram-positive bacteria, protein stability, structured C terminus
Abbreviations: CD, circular dichroism; EMSA, electrophoretic mobility-shoft assay
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() |
---|
Over the past decade, we became interested in the two components regulating the replication of plasmid pIP501: an antisense RNA which acts via transcriptional attenuation (Brantl et al., 1993 ; Brantl & Wagner, 1994
) and the transcriptional repressor CopR (92 aa, 10·6 kDa). CopR represses transcription of the essential repR mRNA (Brantl, 1994
) and prevents convergent transcription of sense and antisense RNAs (Brantl & Wagner, 1997
). Previously, we showed that CopR binds asymmetrically as a preformed dimer at two consecutive major grooves of the DNA (Steinmetzer & Brantl, 1997
; Steinmetzer et al., 1998
) and identified amino acids involved in DNA binding and dimerization (Steinmetzer et al., 2000a
, b
). The last 29 aa of CopR are essential for neither DNA binding nor dimerization, but are essential for protein stability (Kuhn et al., 2000
). The half-life of CopR amounts to 42±5 min. However, whereas C-terminally truncated Cop
20 was, in spite of a drastically shortened half-life, still 100% active in vivo, Cop
24 and Cop
27 retained only 20% activity. Therefore, we focused our interest on the last 20 aa. Circular dichroism (CD)-difference spectra revealed that this sequence is not unstructured, but part of it, most probably between aa 76 and 84 (QVTLELEME) apparently forms a ß-strand with alternating hydrophilic and hydrophobic residues (Kuhn et al., 2000
). This ß-strand forming sequence is also present in the related repressors CopF (Swinfield et al., 1990
) and CopS (Ceglowski & Alonso, 1994
), albeit as QVTLDLEME.
Here, we dissect stabilizing motifs within the C terminus of CopR. Two such motifs were found: the amphiphilic ß-strand sequence that can be replaced by a heterologous ß-strand forming sequence, and the last 7 aa. We demonstrate that neither a region folding predominantly into an -helix nor a completely unstructured region with the same degree of hydrophilicity can completely compensate for a ß-strand.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() |
---|
Construction of Escherichia coli/B. subtilis shuttle vectors containing mutated copR genes.
Plasmids pCOPVT, pCOPVT7 and the plasmids containing individual E to K substitutions were constructed using a single-step PCR on plasmid pCOP7 as template with primer 486-34 (Kuhn et al., 2000
) and one of the mutagenic primers described in Table 1
. The resulting 500 bp PCR fragment was digested with BamHI and inserted into the single BamHI site of plasmid pPR7 (Brantl & Behnke, 1992
).
|
Construction of E. coli vectors for overexpression of mutated copR genes.
A single-step PCR with primer B618-30 (Steinmetzer & Brantl, 1997 ) and (for pQVT) primer 951-31 (Steinmetzer et al., 2000a
) or (for pQPE) primer 8 (Table 1
) was used to amplify the mutated copR genes from the E. coli/B. subtilis shuttle vectors pCOPVT and pCOPPE used as templates. The amplified fragments were cleaved with BamHI/PstI and inserted into expression vector pQE9 (Qiagen). In this way, the mutated copR genes contain 11 additional 5' codons encoding Met-Arg-Gly-Ser-His6-Gly-Ser fused to the second codon of copR (Glu).
Pulsechase experiments.
These were performed as described previously (Kuhn et al., 2000 ). In all cases, 100 µl polyclonal antiserum raised against His6-CopR purified from E. coli were used for the precipitation of mutated CopR proteins from the lysates.
Purification of CopR proteins, determination of protein concentration and electrophoretic mobility-shift assay (EMSA).
Overexpression and purification of the proteins from E. coli were performed as described before (Steinmetzer et al., 1998 ). Protein concentrations were determined by Bradford assays based on calibration curves obtained with the values from acidic hydrolysis of His6-CopR. EMSA was performed as described previously (Steinmetzer & Brantl, 1997
).
CD measurements.
Purified proteins in 50 mM phosphate buffer containing 150 mM NaCl and 50% (w/w) glycerol were used. The CD spectra were measured at room temperature in the range from 195 to 260 nm with a JASCO model 710 spectropolarimeter at a scan speed of 50 nm min-1 and 1 nm resolution. The path length of the cells used was 0·1 mm. The spectra were recorded as a mean of 10 scans. The appropriate buffer baseline spectra were subtracted from the protein spectra. To calculate the mean residue ellipticity, the residue concentration used was obtained by multiplying the molar protein concentrations with the number of residues.
RESULTS AND DISCUSSION
Design and analysis of C-terminally substituted CopR mutants in vivo and in vitro
We aimed to answer the following questions by the analysis of half-life and structure of C-terminally mutated CopR proteins: i) does the C terminus need to be acidic to fulfil its stabilizing function? ii) how important are the seven C-terminal amino acids for CopR stability? and iii) is a ß-strand forming sequence necessary for CopR stability?
Plasmids pCOPE80K, pCOPE82K, pCOPE84K, pCOPE85K and pCOPE86K were constructed and used for the analysis of single substitutions of acidic glutamic acid residues by basic lysine residues. To investigate the importance of the C-terminal 7 aa residues located downstream of the ß-strand forming sequence, pCOPVT7 was constructed. To analyse the importance of the previously found ß-strand forming sequence QVTLELEME between aa 76 and 84 of CopR, plasmid pCOPVT was constructed encoding a CopR protein where this sequence was replaced by QVTVTVTVT, a sequence which Brack & Caille (1978)
had shown that to be present in the ß conformation in solution. To analyse whether CopVT is able to regulate the copy number of pIP501 in B. subtilis as well as wild-type CopR, pCOPEVT, a plasmid expressing both regulatory components, RNAIII and the mutated CopVT, was constructed and used for copy-number determinations, since pCOPVT does not express RNAIII. Plasmid pCOPPE was constructed to find out whether the ß-strand forming sequence can be replaced by a sequence predicted to form a random coil. In all full-length mutants designed here, we tried to maintain the degree of hydrophilicity within the C terminus as much as possible, since R. T. Sauers group demonstrated that hydrophilic C-terminal extensions stabilized proteins more than hydrophobic ones (see Introduction).
All plasmids were transferred into B. subtilis strain DB104 and the corresponding strains were used in pulsechase experiments for half-life determinations (see Table 2). To find out whether CopVT was still able to regulate the replication of pIP501 in B. subtilis, copy-number determinations with DB104(pCOPEVT) containing a mutant plasmid expressing wild-type antisense RNA, the second regulatory component, and mutated CopVT were performed. Plasmid pCOPEVT maintained the wild-type copy-number, indicating that CopVT is functional in vivo.
|
|
The C-terminal 7 aa contribute to CopR stabilization by a factor of approximately 2
Deletion of 7 C-terminal amino acids from CopVT (CopVT7) yielded a 1·6-fold reduction of the half-life (70±5 min). A comparison between the half-lives of CopR and Cop
7 (42±5 and 24±1 min respectively) as well as a comparison between the half-lives of CopVT and CopVT
7 (111·5±17·5 and 70±5 min respectively) shows that the 7 C-terminal amino acids (EKSNDFV) contribute to CopR stability by a factor of 1·61·8. Previously, we had also shown that mutated protein Cop
5, with a half-life of 31±6 min, is moderately less stable than wild-type CopR. Four of the C-terminal 7 aa and two of the C-terminal 5 aa are hydrophilic, which does support the role of hydrophilic amino acids in protecting more hydrophobic regions of the protein against proteolytic attack, as suggested by the publications of R. T. Sauers group (Milla et al., 1993
; Parsell & Sauer, 1989
; Parsell et al., 1990
).
An amphiphilic ß-strand structure within the C terminus stabilizes CopR, whereas an -helix or an unstructured region does not
CD measurements with purified CopVT suggested that this protein with the sequence QVTVTVTVT between aa 76 and 84 which has a higher propensity to form an amphiphilic ß-strand has a wild-type-like structure (Fig. 1a). Pulsechase experiments showed that it is nearly threefold more stable in vivo than the wild-type protein [Table 2
, half-lives of CopR (42±5 min) and CopVT (111·5±17·5 min)]. As shown before, replacement of this sequence by a sequence that folds predominantly into an
-helix (Cop20-K; QVTLKLKMK) significantly destabilized CopR (half-life of 1·6±0·4 min; Kuhn et al., 2000
). At that time, however, it was not clear whether this effect was mainly due to structural reasons or to the exchange of an acidic for a basic C terminus. To prove whether an unstructured region can compensate for the ß-strand forming region, we analysed mutated CopPE protein containing the sequence QVTPEPEPE between aa 76 and 84. CD measurements suggested that this mutant contains a random coil region (Fig. 1
) and pulsechase experiments showed that it was less stable than the wild-type (34±3 min compared with 42±5 min), although not to the extent exerted by Cop20-K. The relatively moderate destabilization of CopPE can be explained by the presence of both the stabilizing C-terminal 7 aa and the decisive E80 within the ß-strand forming sequence, each of which contributes by a factor of
2 to CopR stability (see above). Furthermore, it cannot be excluded completely that one or two of the proline residues can form hydrophobic contacts which are made by leucine or valine residues in the case of wild-type CopR or CopVT, respectively, since for example in 434 repressor N terminus, proline is part of the hydrophobic dimeric interface (Aggarwal et al., 1988
). In contrast, the significant destabilization of Cop20-K seems to be predominantly due to structural alterations (ß-strand into
-helix and random coil): the stabilizing 7 C-terminal aa are present in this mutant and replacement of E85 or E86 by K as present in Cop20-K was found to have a stabilizing rather than a destabilizing effect.
These results indicate that the amphiphilic ß-strand is an important stabilizing motif within the C terminus of CopR and that its substitution by an -helix or an unstructured region with the same degree of hydrophilicity cannot, or can only partly, compensate for its stabilization function.
We suggest that an interaction of the ß-strand forming sequence with amino acids located at the N terminus and/or in the central portion of the protein leads to the stabilization of CopR. Among these contacts, at least one ionic contact between E80 and a basic residue should be expected, whereas the other contacts have to be hydrophobic ones involving one or more of the hydrophobic aa within the ß-strand (V77, L79, L81 or M83). Experiments are under way to prove this hypothesis.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() |
---|
Bowie, J. U. & Sauer, R. T. (1989). Identification of C-terminal extensions that protect proteins from intracellular proteolysis. J Biol Chem 264, 7596-7602.
Brack, A. & Caille, A. (1978). Synthesis and beta-conformation of copolypeptides with alternating hydrophilic and hydrophobic residues. Int J Pept Protein Res 11, 128-139.[Medline]
Brantl, S. (1994). The copR gene product of plasmid pIP501 acts as a transcriptional repressor at the essential repR promoter. Mol Microbiol 14, 473-483.[Medline]
Brantl, S. & Behnke, D. (1992). Copy-number control of the streptococcal plasmid pIP501 occurs at three levels. Nucleic Acids Res 20, 395-400.[Abstract]
Brantl, S. & Wagner, E. G. H. (1994). Antisense RNA-mediated transcriptional attenuation occurs faster than stable antisense/target RNA pairing: an in vitro study of plasmid pIP501. EMBO J 13, 3599-3607.[Abstract]
Brantl, S. & Wagner, E. G. H. (1997). Dual function of the copR gene product of plasmid pIP501. J Bacteriol 179, 7016-7024.[Abstract]
Brantl, S., Birch-Hirschfeld, E. & Behnke, D. (1993). RepR protein expression on plasmid pIP501 is controlled by an antisense RNA-mediated transcription attenuation mechanism. J Bacteriol 175, 4052-4061.[Abstract]
Ceglowski, P. & Alonso, J. C. (1994). Gene organization of the Streptococcus pyogenes plasmid pDB101: sequence analysis of the orfcopS region. Gene 145, 33-39.[Medline]
Gomis-Rüth, F. X., Sola, M., Acebo, P. & 7 other authors (1998). The structure of plasmid-encoded transcriptional repressor CopG unliganded and bound to its operator. EMBO J 17, 74047415.
Kuhn, K., Steinmetzer, K. & Brantl, S. (2000). Transcriptional repressor CopR: the structured acidic C terminus is important for protein stability. J Mol Biol 300, 1021-1031.[Medline]
Milla, M. E., Brown, M. B. & Sauer, R. T. (1993). P22 Arc repressor: enhanced expression of unstable mutants by addition of polar C-terminal sequences. Protein Sci 2, 2198-2205.
Milla, M. E., Brown, M. B. & Sauer, R. T. (1994). Protein stability effects of a complete set of alanine substitutions in Arc repressor. Nat Struct Biol 1, 518-523.[Medline]
Parsell, D. A. & Sauer, R. T. (1989). The structural stability of a protein is an important determinant of its proteolytic susceptibility in Escherichia coli. J Biol Chem 264, 7590-7595.
Parsell, D. A., Silber, K. R. & Sauer, R. T. (1990). Carboxy-terminal determinants of intracellular protein degradation. Genes Dev 4, 277-286.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[Abstract]
Steinmetzer, K. & Brantl, S. (1997). Plasmid pIP501 encoded transcriptional repressor CopR binds asymmetrically at two consecutive major grooves of the DNA. J Mol Biol 269, 684-693.[Medline]
Steinmetzer, K., Behlke, J. & Brantl, S. (1998). Plasmid pIP501 encoded transcriptional repressor CopR binds to its target DNA as a dimer. J Mol Biol 283, 595-603.[Medline]
Steinmetzer, K., Hillisch, A., Behlke, J. & Brantl, S. (2000a). Transcriptional repressor CopR: structure model based localization of the DNA binding motif. Proteins 38, 393-406.[Medline]
Steinmetzer, K., Hillisch, A., Behlke, J. & Brantl, S. (2000b). Transcriptional repressor CopR: amino acids involved in forming the dimeric interphase. Proteins 39, 408-416.[Medline]
Swinfield, T.-J., Oultram, J. D., Thompson, E. E., Brehm, J. K. & Minton, N. P. (1990). Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAMß1. Gene 87, 79-90.[Medline]
Received 23 May 2001;
revised 17 August 2001;
accepted 20 August 2001.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |