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Originally published In Press as doi:10.1074/jbc.M413596200 on February 16, 2005

J. Biol. Chem., Vol. 280, Issue 16, 16151-16156, April 22, 2005
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The Crucial Role of Conserved Intermolecular H-bonds Inaccessible to the Solvent in Formation and Stabilization of the TL5·5 SrRNA Complex*

George M. Gongadze{ddagger}§, Alexey P. Korepanov{ddagger}, Elena A. Stolboushkina{ddagger}, Natalia V. Zelinskaya{ddagger}, Anna V. Korobeinikova{ddagger}, Maxim V. Ruzanov{ddagger}, Boris D. Eliseev{ddagger}, Oleg S. Nikonov{ddagger}, Stanislav V. Nikonov{ddagger}, Maria B. Garber{ddagger}, and Valery I. Lim||

From the {ddagger}Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia and the ||Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, Moscow, 119991 Russia

Received for publication, December 2, 2004 , and in revised form, February 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of the structures of two complexes of 5 S rRNA with homologous ribosomal proteins, Escherichia coli L25 and Thermus thermophilus TL5, revealed that amino acid residues interacting with RNA can be divided into two different groups. The first group consists of non-conserved residues, which form intermolecular hydrogen bonds accessible to solvent. The second group, comprised of strongly conserved residues, form intermolecular hydrogen bonds that are shielded from solvent. Site-directed mutagenesis was used to introduce mutations into the RNA-binding site of protein TL5. We found that replacement of residues of the first group does not influence the stability of the TL5·5 S rRNA complex, whereas replacement of residues of the second group leads to destabilization or disruption of the complex. Stereochemical analysis shows that the replacements of residues of the second group always create complexes with uncompensated losses of intermolecular hydrogen bonds. We suggest that these shielded intermolecular hydrogen bonds are responsible for the recognition between the protein and RNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abundant structural information on RNA·protein complexes has appeared during last five years (see for review Refs. 1–4). Thorough analysis of this information should contribute fundamentally toward understanding the principles of RNA· protein recognition. In the present work the structures of two complexes of 5 S rRNA with homologous ribosomal proteins, Escherichia coli L25 (5) (PDB code 1DFU [PDB] ) and Thermus thermophilus TL5 (6) (PDB code 1FEU [PDB] ), have been analyzed.

L25 and TL5 belong to the so-called CTC family of bacterial proteins. The first protein described under the CTC abbreviation was a general stress protein of Bacillus subtilis (7). Later the genes for homologous proteins were found in most of the sequenced bacterial genomes (8, 9). Among these proteins there are one-domain, two-domain, and even three-domain representatives (5, 6, 10, 11); all of them have a domain homologous to E. coli 5 S rRNA-binding ribosomal protein L25, which consists of only one domain (11). Beside L25, two other proteins of the CTC family were found in bacterial ribosomes, TL5 of T. thermophilus (12) and CTC of Deinococcus radiodurans (10). E. coli L25, T. thermophilus TL5, and D. radiodurans CTC specifically bind 5 S rRNA at the same site, the so-called loop E region (10, 13, 14). The stress protein CTC of B. subtilis was also shown to bind to the same site on 5 S rRNA (15). It was suggested that all CTC proteins could bind 5 S rRNA through a domain homologous to E. coli L25 (6).

T. thermophilus ribosomal protein TL5 was discovered and investigated thoroughly by our group (12, 14, 16). It is a two-domain protein. Its RNA-binding N-terminal domain is homologous to E. coli L25. The isolated N-terminal fragment of TL5 (NfrTL5)1 possesses the same 5 S rRNA binding ability as the full-size protein (14). The crystal structures of E. coli L25 and T. thermophilus TL5 complexed with virtually the same fragment of E. coli 5 S rRNA (Fig. 1) have been determined at a high resolution (5, 6). Despite rather low sequence identity between E. coli L25 and the N-terminal domain of T. thermophilus TL5 (18%), their three-dimensional structures and their modes of interaction with 5 S rRNA are very similar. Among the amino acid residues that form hydrogen bonds with RNA in the L25·5 S rRNA and TL5·5 S rRNA complexes, only five are strongly conserved in CTC family proteins (Arg-10, Arg-19, Tyr-29, His-85 and Asp-87, TL5 nomenclature). Superposition of the structures of the L25·5 S rRNA and TL5·5 S rRNA complexes reveals that the conformations of the side chains of the conserved residues are practically the same in the two complexes (6). Moreover, their contacts with RNA also coincide. These observations allow us to suggest that the conserved residues form an RNA recognition module on the protein surface (Fig. 1). This hypothesis stimulated us to use site-directed mutagenesis to change the residues involved in RNA binding in the TL5·5 S rRNA complex. We have found that replacement of the strongly conserved residues leads to destabilization or disruption of the complex. Analysis shows that intermolecular hydrogen bonds formed by these residues are inaccessible to the solvent and that substitutions of these residues cause the uncompensated loss of one or more intermolecular hydrogen bonds. We suggest that these shielded intermolecular hydrogen bonds are responsible for the recognition between the protein and RNA.



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  		FIG. 1.
Comparison of the structures of proteins TL5 and L25 complexed with 5 S rRNA. A, general view. The five conserved residues are shown as metallic spheres. B, superposition of RNA-recognition modules of TL5 (blue) and L25 (red).

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis—To prepare wild type and mutant forms of the NfrTL5 polypeptide, site-directed mutagenesis was carried out by PCR using oligonucleotides that contained the desired mutations. The ntl5WT and mutant fragments of the gene (ntl5H85A, ntl5H85N, ntl5H85T, ntl5H85F, ntl5D87N, ntl5D87E, and ntl5D87S) were amplified from pET11c/TL5 DNA by PCR. We used forward primer WT, 5'-AAATTGAAGGAGATATACATATGGAGTAC-3' and reverse primers, WT, 5'-GGGCGGATCCTAGAGGACGAAGAAGTCC-3'; H85A, 5'-GGGCGGATCCTAGAGGACGAAGAAGTCCACCGCCTCGGGGC-3'; H85N, 5'-ATATGGATCCTAGAGGACGAAGAAGTCCACGTTCTCGGGGC-3'; H85T, 5'-ATATGGATCCTAGAGGACAAAAAAGTCCACGGTCTCGGGGC-3'; H85F, 5'-ATATGGATCCTAGAGGACGAAGAAGTCCACGAACTCGGGGC-3'; D87N, 5'-ATATGGATCCTAGAGGACGAAGAAGTTAACGTGCTC-3'; D87E, 5'-ATATGGATCCTAGAGGACGAAGAATTCCACGTGC-3'; and D87S, 5'-ATATGGATCCTAGAGGACGAAGAAGGACACGTGCTC-3'. The ntl5R10A, ntl5R19A, ntl5Y29F, ntl5Y29S, and ntl5Y29R mutant fragments were constructed in two steps. In step 1, fragments carrying a mutation were amplified from pET11c/TL5 DNA by PCR with a T7 forward primer and corresponding oligonucleotides for each fragment, R10A, 5'-CTCCCTCCGCGTAGTAC-3'; R19A, 5'-GCCCGCCCGCGCGAGGGCC-3'; Y29F, 5'-GGTTGAACATGAGCCCGGGGAGC-3'; Y29S, 5'-GGTTGGACATGAGCCCCGGGAGC-3'; and Y29R, 5'-GCCGGTTGCGCATGAGCCC-3'. In step 2, the entire ntl5 fragments were then amplified from pET11c/TL5 DNA using the PCR products obtained in step 1 and the reverse primer WT. To construct the ntl5H85A mutant, the gene fragment was amplified in step 1 using 5'-GCGCCCCGAGGCGGTGGAC and the T7 reverse primer. The entire ntl5 fragment was then completed by PCR using the forward primer WT. The final PCR products were digested with NdeI and BamHI and cloned into the corresponding sites of pET11c (17). All plasmids were verified by sequencing.

Gene Expression and Recombinant Protein Purification—All the mutant proteins were expressed in E. coli BL21(DE3) pLysS (Stratagene) using the Studier expression system (18). Two liters of 1% LB medium containing 100 µg/ml ampicillin and 30 µg/ml chloramphenicol were incubated at 37 °C with vigorous shaking until the A590 nm reached ~0.7. Expression was induced by adding 1 mM isopropyl-{beta}-D-thiogalactopyranoside. After 3 h, the cells were harvested by centrifugation (6000 x g for 15 min) and kept frozen at –20 °C. Three grams of cells were suspended in 20 ml of HS buffer (50 mM Tris-HCl, pH 7.5, 1.2 M NaCl, 20 mM MgCl2, and 5 mM {beta}-mercaptoethanol) and disrupted by ultrasonic treatment. After centrifugation (14,000 x g for 30 min followed by 400,000 x g for 1 h), the cleared cell extract was diluted 10-fold with HS buffer containing no NaCl or MgCl2 and loaded onto a CM-Sepharose FF (Amersham Biosciences) column. The protein was eluted using a linear NaCl concentration gradient (200–800 mM). Protein purity was assessed by SDS-PAGE (19).

Obtaining and Radioactive Labeling of E. coli 5 S rRNA—Ribosomal RNA was isolated from E. coli ribosomes as described previously (20) and separated by gel filtration on Sephadex G-150 (Amersham Biosciences). 5'-end labeling using [{gamma}-32P]ATP and T4 polynucleotide kinase was carried out as described previously (21). Labeled RNA was purified by electrophoresis on 8% polyacrylamide gels (acrylamide/N,N'-methylene-bisacrylamide 19:1, w/w) containing 8 M urea.

Gel-shift Analysis—Gel-shift analysis was carried out as described earlier (22). A sample of E. coli 5 S rRNA was incubated in TKM buffer (20 mM Tris-HCl, pH 7.5, 200 mM KCl, 10 mM MgCl2) for 2 min at 60 °C and chilled to room temperature. Then, an aliquot of the RNA (3–4 µg) was incubated in TKM buffer with the indicated amount of NfrTL5 for 10 min at 37 °C. Concentrations of 5 S rRNA and NfrTL5 proteins in the incubation mixture were 1 x 10–6–1 x 10–5M and 1 x 10–6–1 x 10–4 M, respectively. The reaction mixtures were loaded onto a 12% (w/v) polyacrylamide gel (70 x 80 x 0.75 mm, acrylamide/N,N'-methylenebisacrylamide 19:1, w/w) containing TAM buffer (90 mM Tris acetate, pH 7.8, 10 mM MgCl2) and run at 90 V at room temperature until xylene cyanol started to leave the gel. After the procedure the gels were stained with 0.25% (w/v) toluidine blue in 5% acetic acid, 20% ethanol and washed with water until the background stain disappeared.

Formation of RNA·Protein Complexes and Filter Binding Assay—The procedures were carried out as described previously (23). E. coli 5'-32P-5 S rRNA was heated at 60 °C for 2 min in the buffer for complex formation (50 mM Tris-HCl, pH 7.5, 170 mM KCl, 10 mM MgCl2) and cooled to room temperature. Then, aliquots (3000–5000 cpm, quantified by scintillation counting) of the RNA and NfrTL5 were mixed in the same buffer containing 20 µg/ml bovine serum albumin and incubated for 30 min on ice. The incubation volume was 50 µl, and the concentration of the protein varied from 1 x 10–10 M to 3 x 10–5M. Each assay was done in duplicate. The concentration of the labeled RNA in the assay was negligible (<1 x 10–9). Protein·RNA complexes were retained by filtration at 4 °C on nitrocellulose membranes (Millipore GS, 0.22µ m). The filters were washed once with 350 µl of the ice-cold buffer containing 20 µg/ml bovine serum albumin, dried, and analyzed for radioactivity. Nonspecific retention of RNA (4–6%) was measured by filtering the complete reaction mixture in the absence of NfrTL5. Specific retention (total retention minus nonspecific retention) was generally 30–60%. The apparent dissociation constants (Kd) correspond to the concentration of NfrTL5 required to obtain half-saturation (assuming that complex formation obeys a simple bimolecular equilibrium) and were calculated according to the equation, {Theta} = [NfrTL5]/(Kd + [NfrTL5]), where {Theta} represents the fraction of radiolabeled RNA bound to the filter.

RNA and Protein Sequences Alignment, Structural Modeling of the Mutations—Protein sequences were obtained from the NCBI GenBankTM(8) database and Pedant (9) database and aligned using PSI-PLAST (24). The 5 S ribosomal RNA database (25) was used as a source of 5 S rRNA sequences. Comparative analysis of the L25 and TL5 structures and structural modeling of mutations in TL5 were made by program O (26). To determine the accessibility of atoms forming intermolecular hydrogen bonds between protein and 5 S rRNA, the Areaimol program was used (27). The radius of a water molecule was taken as equivalent to 1.4 Å.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Mutations on TL5·5 S rRNA Interactions—In all experiments we used the NfrTL5 (residues 1–91), because in the isolated state this fragment possesses the same 5 S rRNA binding ability as the full-size TL5 (14). The five strongly conserved amino acid residues, as well as some non-conserved residues that bind RNA, were changed by site-directed mutagenesis. Non-conserved residues were replaced only by alanine to eliminate hydrogen bonding with RNA. In the case of the conserved residues, a variety of different mutations were introduced. The mutant NfrTL5 polypeptides were isolated from overproducing strains and purified by the procedure described earlier (14, 22). The 5 S rRNA binding ability of the mutants was tested by gel-shift and filter binding assays. There is a good agreement between results obtained by these two techniques. All results are summarized in Table I.


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  		TABLE I
RNA-binding ability of the mutant N-terminal fragments of TL5 protein

 
Fig. 2A shows a selection of NfrTL5·5 S rRNA-binding curves. It can be seen that a plateau (saturation area) in binding curves is reached at 1–10 mM protein concentration region. To test the stability of 5 S rRNA·NfrTL5 complexes in this protein concentration region, we used the gel-shift method (a typical gel-shift pattern is shown in Fig. 2B). Stable complex of wild type NfrTL5·5 S rRNA was observed at 1–2 mM protein concentration (Fig. 2B, lane 2). In case of mutations of nonconserved residues involved in 5 S rRNA binding (Lys-14, Ser-16, and Arg-20), the same protein concentration was enough to form stable complexes between 5 S rRNA and the mutant NfrTL5 polypeptides (Table I). Even NfrTL5, which bears the double mutation S16A/R20A, has an RNA-binding curve virtually identical to that for wild type NfrTL5 (Fig. 2A). Thus, mutations of the non-conserved residues do not influence the RNA binding ability of the protein.



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  		FIG. 2.
5 S rRNA-binding properties of NfrTL5 and its mutant forms. Details of the assay conditions are described under "Materials and Methods." A, saturation-binding curves with 32P-labeled 5 S rRNA E. coli. B, gel-shift analysis of formation of some NfrTL5·5 S rRNA complexes. Concentration of the RNA was 1 µM (lane 2) and 10 µM (lanes 3–7). Lane 1, 5 S rRNA only; lane 2, +NfrTL5 wild type (1.2 µM); lane 3, +NfrTL5 D87E (30 µM); lane 4, +NfrTL5 D87E (70 µM); lane 5, +NfrTL5 H85T (25 µM); lane 6, +NfrTL5 R10A (25 µM); lane 7, +NfrTL5 Y29F (50 µM). The protein concentrations in the incubation mixture are shown in parentheses.

 
In contrast, mutations of the five strongly conserved residues (Arg-10, Arg-19, Tyr-29, His-85, and Asp-87) lead to destabilization or disruption of the NfrTL5·5 S rRNA complex in all cases. Replacement of one of the three strongly conserved residues (Arg-10, Arg-19, or His-85) leads to 10-fold Kd increase (Table I and Fig. 2A). Complexes of these mutants with the RNA could not be formed at protein concentrations lower than 20–30 mM (Fig. 2B, lanes 5 and 6). Moreover, the bands of these complexes have tails of free RNA. We suggest that this tailing is a consequence of the lower stability of the complexes between the mutant proteins and 5 S rRNA. Thus, mutations of these three strongly conserved residues markedly destabilize the complex.

The most dramatic effect was observed for mutations of Tyr-29 or Asp-87 (Table I). Excluding D87E, all mutations of these two residues lead to disruption of the complex (no visible retention of labeled RNA at nitrocellulose membrane at protein concentration 5 mM). NfrTL5 bearing the Y29F mutation could not form a complex with 5 S rRNA even at 50 mM protein concentration (Fig. 2B, lane 7). The D87E mutation does not disrupt the complex but destabilizes it very strongly (Table I; Fig. 2, A and B, lanes 3 and 4). Besides making buried intermolecular H-bonds with RNA, the side chains of Tyr-29 and Asp-87 form an H-bond with one another. This H-bond stabilizes the conformations of the side chains of these two residues at the protein surface. All replacements of Tyr-29 and Asp-87 that we tested, excluding D87E, not only eliminate the intermolecular H-bonds between these residues and RNA but also disrupt the H-bond between these two residues. As for D87E, modeling shows that glutamic acid could make H-bonds with Tyr-29 and the RNA molecule, but the significantly larger size of the glutamic acid side chain creates some steric hindrance to tight protein-RNA contact. Thus, our data show that any change of side chains of residues located at positions 29 and 87 (specifically, the displacement of the atoms that make H-bonds with the rRNA) is critical for the specific recognition of 5 S rRNA by protein TL5.

Naturally Occurring Changes in CTC Proteins—Naturally occurring changes of the five strongly conserved amino acid residues in CTC proteins shed further light on their role in RNA binding. Analysis of 150 homologous proteins of CTC family (8, 9) reveals only 1 replacement for Tyr-29 and <10 replacements for Asp-87 (Table II). One of the natural changes is the substitution of aspartic acid by glutamic acid at the position corresponding to Asp-87 in protein TL5. This substitution was found in the members of Enterococcus genus. According to our results, such a substitution strongly decreases the 5 S rRNA binding ability of protein TL5 (Table I). However, in bacterial species where the Asp/Glu exchange is observed at the corresponding positions of other CTC sequences, there is an interesting change in the sequence of the 5 S rRNA (25). Fig. 3 shows the sequence of the loop E region of 5 S rRNA from Enterococcus faecalis. In comparison with E. coli 5 S rRNA, there is the only change, in E. faecalis 5 S rRNA G75 is replaced by U. In the TL5·5 S rRNA complex, G75 forms an H-bond with Asp-87. This appears to be an example of co-evolution of the structures of two interacting macromolecules. This assumption can be tested experimentally; the D87E mutant should form a stable complex with E. coli 5 S rRNA if G75 is replaced by U.


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  		TABLE II
Changes of the strongly conserved amino acid residues in proteins of the CTC family

 



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  		FIG. 3.
Schemes of the secondary structure of the 5 S rRNA E-loop region from various bacterial species. Conserved nucleotides are black (>80% in the bacterial 5 S rRNA), non-conserved nucleotides are gray. The arrow indicates the nucleotide that interacts with Asp-87 of the TL5 protein. Changes in conserved nucleotides are shown in open typeface. Replacements of Asp-87 in the corresponding CTC proteins are shown.

 
Another interesting case is the naturally occurring replacement of Asp-87 by Ser or Ala found in CTC protein sequences of Cyanobacteria. Such mutations lead to disruption of the TL5·5 S rRNA complex (Table I). However, there are many changes in the 5 S rRNA of Cyanobacteria (14). The secondary structure of the loop E region of cyanobacterial 5 S rRNA is most likely different from that in the majority of the known bacterial 5 S rRNA structures (Fig. 3). This may also represent an example of co-evolution.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A structural analysis of the TL5·5 S rRNA complex shows that the contacting sites of TL5 and 5 S rRNA are remarkably complementary (6). They are rather flat regions, a four-stranded {beta}-sheet with adjacent parts of the N-terminal {beta}-strand and {alpha}-helix in the protein and an irregular shallow groove in the RNA helix. The strongly conserved residues, Arg-10, Arg-19, and His-85, and the non-conserved residues, Lys-14, Arg-20, Arg-31, Lys-36, Arg-72, and Lys-78, provide a positively charged protein surface. The negative charge in the RNA-contacting region results from a specific disposition of phosphate groups. Amino acid residues involved in RNA binding in the TL5·5 S rRNA complex can be subdivided into two groups, the first group is non-conserved residues, and the second group is strongly conserved residues. The strongly conserved residues (Arg-10, Arg-19, Tyr-29, His-85, and Asp-87) are situated in the central part of the RNA-contacting surface of TL5, and the non-conserved residues are located at the periphery of this surface (Fig. 4).



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  		FIG. 4.
A view of the protein TL5 surface from the 5 S rRNA side. The contact regions are shown in bright gray. The RNA-binding residues are labeled. The strongly conserved residues are indicated by rectangular frames, and non-conserved residues are indicated by ellipsoidal frames.

 
In the present work, a comparative structural analysis of two homologous complexes, TL5·5 S rRNA and L25·5 S rRNA, revealed that in both complexes the intermolecular hydrogen bonds of the non-conserved residues are accessible to the solvent, whereas the five strongly conserved residues and their intermolecular hydrogen bonds are shielded from the solvent (Table III). The site-directed mutagenesis of TL5 shows that replacement of residues of the first group does not influence the stability of the TL5·5 S rRNA complex, whereas a replacement of residues of the second group leads to destabilization or disruption of the complex.


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  		TABLE III
Protein-RNA hydrogen bonds in the TL5·5 S rRNA complex

Length of a hydrogen bond and accessibility of atoms forming this hydrogen bond are determined as the mean of corresponding values measured for two complexes in the asymmetric unit.

 
Thanks to the accessibility of the non-conserved residues and their RNA partners to solvent, the intermolecular hydrogen bonds of these residues can be disrupted and replaced by new hydrogen bonds formed with solvent molecules. This means that the RNA·protein complex cannot be strongly destabilized by disruption of intermolecular hydrogen bonds formed by the non-conserved amino acid residues. Such substitutions may not be completely neutral, because they may cause entropic changes, but such differences among residue side chains are very small and do not greatly affect complex stability. Substitutions could also affect other non-covalent interactions such as van der Waals interactions, but, again, changes caused by such other non-covalent interactions (28) are on the order of the thermal energy kT (~2.5 kJ/mol). Therefore, the mutations of the solvent-exposed non-conserved amino acid residues do not cause the dissociation of the RNA·protein complex (Table I).

A different situation arises with intermolecular hydrogen bonds formed by conserved amino acid residues. These residues and their intermolecular hydrogen bonds are completely or partially inaccessible to the solvent. For this reason, the disruption of an intermolecular hydrogen bond of a conserved residue cannot be compensated by the formation of new hydrogen bonds. The uncompensated loss of a single intermolecular hydrogen bond should destabilize the RNA·protein complex by 20 ± 5 kJ/mol. Moreover, because displacements of the bonding groups by just an angstrom or so would compromise them, these intermolecular hydrogen bonds anchor the complex. All of this explains why the mutations in conserved residues that disrupt intermolecular hydrogen bonds cause significant destabilization or full disruption of the complex.

It is typical that conserved intermolecular hydrogen bonds in the complexes of ribosomal proteins with their specific targets on rRNAs are not accessible to the solvent. This observation led us to the conclusion that the shielded intermolecular hydrogen bonds are a major factor in recognition. The results obtained in this work are in good agreement with our previous findings on the organization of RNA-recognition modules on the surface of ribosomal proteins (29).


   FOOTNOTES
 
* This work was supported in part by the Russian Academy of Sciences (RAS), the Russian Foundation for Basic Research (03-04-48393, 04-04-49634 and 02-04-48539), the Council at the Russian Federation President (Grant for outstanding scientific schools 1969.2003.4.), and a grant from the Program of Molecular and Cellular Biology RAS (10002-251/-10/145-161/140503-085). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence may be addressed. Tel.: 967-73-27-70; Fax: 95-632-78-71; E-mail: gongadze{at}vega.protres.ru. ¶ Supported by International Research Scholar's award from the Howard Hughes Medical Institute. To whom correspondence may be addressed. Tel.: 967-73-27-70; Fax: 95-632-78-71; E-mail: garber{at}vega.protres.ru.

1 The abbreviations used are: NfrTL5, N-terminal fragment of TL5; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank N. A. Matveeva for technical assistance, R. A. Zimmermann for critical consideration of the paper, and J. F. Curran for the editing of the text.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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