Simultaneous and functional binding of SmpB and EF-Tu-TP to the alanyl acceptor arm of tmRNA.

Transfer-messenger RNA (tmRNA) mimics functions of aminoacyl-tRNA and mRNA, subsequently, when rescuing stalled ribosomes on a 3' truncated mRNA without stop codon in bacteria. In addition, this mechanism marks prematurely terminated proteins by a C-terminal peptide tag as a signal for degradation by specific cellular proteases. For Escherichia coli, previous studies on initial steps of this "trans-translation" mechanism revealed that tmRNA alanylation by Ala-tRNA synthetase and binding of Ala-tmRNA by EF-Tu-GTP for subsequent delivery to stalled ribosomes are inefficient when compared to analogous reactions with canonical tRNA(Ala). In other studies, protein SmpB and ribosomal protein S1 appeared to bind directly to tmRNA and to be indispensable for trans-translation. Here, we have searched for additional and synergistic effects of the latter two on tmRNA alanylation and its subsequent binding to EF-Tu-GTP. Kinetic analysis of functioning combined with band-shift experiments and structural probing demonstrate, that tmRNA may indeed form a multimeric complex with SmpB, S1 and EF-Tu-GTP, which leads to a considerably enhanced efficiency of the initial steps of trans-translation. Whereas S1 binds to the mRNA region of tmRNA, we have found that SmpB and EF-Tu both interact with its acceptor arm region. Interaction with SmpB and EF-Tu was also observed at the acceptor arm of Ala-tRNA(Ala), but there the alanylation efficiency was inhibited rather than stimulated by SmpB. Therefore, SmpB may function as an essential modulator of the tRNA-like acceptor arm of tmRNA during its successive steps in trans-translation.

Protein factors associated with the SsrA.SmpB tagging and ribosome rescue complex.

SsrA RNA acts as a tRNA and mRNA to modify proteins whose synthesis on ribosomes has stalled. Such proteins are marked for degradation by addition of peptide tags to their C termini in a reaction mediated by SsrA RNA and SmpB, a specific SsrA-RNA binding protein. Evidence is presented here for the existence of a larger ribonucleoprotein complex that contains ribosomal protein S1, phosphoribosyl pyrophosphate synthase, RNase R, and YfbG in addition to SsrA RNA and SmpB. Biochemical, genetic, and phylogenetic results suggest potential roles for some of these factors in various stages of the ribosome rescue and tagging process and/or the presence of functional interactions between one or more of these proteins and SsrA.

The SsrA-SmpB system for protein tagging, directed degradation and ribosome rescue.

Bacteria contain a remarkable RNA molecule - known alternatively as SsrA RNA, tmRNA, or 10Sa RNA - that acts both as a tRNA and as an mRNA to direct the modification of proteins whose biosynthesis has stalled or has been interrupted. These incomplete proteins are marked for degradation by cotranslational addition of peptide tags to their C-termini in a reaction that is mediated by ribosome-bound SsrA RNA and an associated protein factor, SmpB. This system plays a key role in intracellular protein quality control and also provides a mechanism to clear jammed or obstructed ribosomes. Here the structural, functional and phylogenetic properties of this unique RNA and its associated factors are reviewed, and the intracellular proteases that act to degrade the proteins tagged by this system are also discussed.

SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA).

In bacteria, SsrA RNA recognizes ribosomes stalled on defective messages and acts as a tRNA and mRNA to mediate the addition of a short peptide tag to the C-terminus of the partially synthesized nascent polypeptide chain. The SsrA-tagged protein is then degraded by C-terminal-specific proteases. SmpB, a unique RNA-binding protein that is conserved throughout the bacterial kingdom, is shown here to be an essential component of the SsrA quality-control system. Deletion of the smpB gene in Escherichia coli results in the same phenotypes observed in ssrA-defective cells, including a variety of phage development defects and the failure to tag proteins translated from defective mRNAs. Purified SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo. Formation of an SmpB-SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain. SsrA RNA is present at wild-type levels in the smpB mutant arguing against a model of SsrA action that involves direct competition for transcription factors.

Origins of DNA-binding specificity: role of protein contacts with the DNA backbone.

A central question in protein-DNA recognition is the origin of the specificity that permits binding to the correct site in the presence of excess, nonspecific DNA. In the P22 Arc repressor, the Phe-10 side chain is part of the hydrophobic core of the free protein but rotates out to pack against the sugar-phosphate backbone of the DNA in the repressor-operator complex. Characterization of a library of position 10 variants reveals that Phe is the only residue that results in fully active Arc. One class of mutants folds stably but binds operator with reduced affinity; another class is unstable. FV10, one member of the first class, binds operator DNA and nonoperator DNA almost equally well. The affinity differences between FV10 and wild type indicate that each Phe-10 side chain contributes 1.5-2.0 kcal to operator binding but less than 0.5 kcal/mol to nonoperator binding, demonstrating that contacts between Phe-10 and the operator DNA backbone contribute to binding specificity. This appears to be a direct contribution as the crystal structure of the FV10 dimer is similar to wild type and the Phe-10-DNA backbone interactions are the only contacts perturbed in the cocrystal structure of the FV10-operator complex.

A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein.

The DnaK and DnaJ heat shock proteins function as the primary Hsp70 and Hsp40 homologues, respectively, of Escherichia coli. Intensive studies of various Hsp70 and DnaJ-like proteins over the past decade have led to the suggestion that interactions between specific pairs of these two types of proteins permit them to serve as molecular chaperones in a diverse array of protein metabolic events, including protein folding, protein trafficking, and assembly and disassembly of multisubunit protein complexes. To further our understanding of the nature of Hsp70-DnaJ interactions, we have sought to define the minimal sequence elements of DnaJ required for stimulation of the intrinsic ATPase activity of DnaK. As judged by proteolysis sensitivity, DnaJ is composed of three separate regions, a 9-kDa NH2-terminal domain, a 30-kDa COOH-terminal domain, and a protease-sensitive glycine- and phenylalanine-rich (G/F-rich) segment of 30 amino acids that serves as a flexible linker between the two domains. The stable 9-kDa proteolytic fragment was identified as the highly conserved J-region found in all DnaJ homologues. Using this structural information as a guide, we constructed, expressed, purified, and characterized several mutant DnaJ proteins that contained either NH2-terminal or COOH-terminal deletions. At variance with current models of DnaJ action, DnaJ1-75, a polypeptide containing an intact J-region, was found to be incapable of stimulating ATP hydrolysis by DnaK protein. We found, instead, that two sequence elements of DnaJ, the J-region and the G/F-rich linker segment, are each required for activation of DnaK-mediated ATP hydrolysis and for minimal DnaJ function in the initiation of bacteriophage lambda DNA replication. Further analysis indicated that maximal activation of ATP hydrolysis by DnaK requires two independent but simultaneous protein-protein interactions: (i) interaction of DnaK with the J-region of DnaJ and (ii) binding of a peptide or polypeptide to the polypeptide-binding site associated with the COOH-terminal domain of DnaK. This dual signaling process required for activation of DnaK function has mechanistic implications for those protein metabolic events, such as polypeptide translocation into the endoplasmic reticulum in eukaryotic cells, that are dependent on interactions between Hsp70-like and DnaJ-like proteins.

Regulation of the synthesis and secretion of transferrin and cyclic protein-2/cathepsin L by mature rat Sertoli cells in culture.

While numerous studies have examined the response of immature rat Sertoli cells to specific hormones and growth factors, the regulation of mature cells in vitro has not been well examined because highly purified cells have been difficult to isolate. We now describe a detailed method for isolating Sertoli cells from mature (> 60 days of age) rats and generating primary cultures of these cells greater than 90% in purity. We demonstrate that cell density, hormones, and growth factors regulate the synthesis or secretion of two Sertoli cell products, transferrin and Cyclic Protein-2 (CP-2)/cathepsin L. Cell density modulated the response of mature Sertoli cells to some hormones; insulin (at 10 micrograms/ml) and epidermal growth factor (EGF) acted synergistically to stimulate transferrin synthesis by 80% when cells were cultured at a density of 1.65 x 10(5) cells/cm2 but had no effect on transferrin synthesis by cells cultured at 1.46 x 10(5) cells/cm2. A mixture of FSH, retinol, and testosterone increased transferrin synthesis by 30% at both cell densities, and this stimulation was independent of the effect of EGF and insulin. CP-2/cathepsin L synthesis was significantly stimulated by increased cell density. FSH, retinol, and testosterone also stimulated CP-2/cathepsin L synthesis by 30%; however, this stimulation just missed being statistically significant. Finally, we demonstrated that secretion of transferrin and CP-2 was reduced when cells were cultured in the presence of interleukin-1 alpha, a cytokine synthesized by Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Germ cell-Sertoli cell interactions: the effect of testicular maturation on the synthesis of cyclic protein-2 by rat Sertoli cells.

Cyclic Protein-2 (CP-2) is synthesized in a stage-specific manner by mature rat Sertoli cells within stage VI and VII seminiferous tubules. To determine how testicular maturation affects CP-2 synthesis, we cultured 20 cm of tubules encompassing all stages of the cycle from rats 17, 35, 45, and 75 days old. The greatest increase in CP-2 synthesis was found to occur between 35 and 45 days and exceeded that observed for transferrin and sulfated glycoprotein (SGP)-2. Additionally, two-dimensional gel analysis indicated that secretion of CP-2 increased from 35 to 45 days to a greater extent than the secretion of SGP-1 and SGP-2 and transferrin. Biochemical analysis also demonstrated that CP-2 synthesis was stage-specific by 45 days. Immunocytochemistry expanded these observations; CP-2 was not detected in 7-35-day-old Sertoli cells. However, at 36 days, CP-2 was detected in Sertoli cells in stage VI and VII tubules but not at any other stage. CP-2 concentration in stage VI-VII tubules was increased by 38 days, but was unchanged thereafter. Finally, we immunocytochemically examined age-related changes in CP-2 concentration of the proximal convoluted kidney tubule. This analysis revealed that, at 1 wk, CP-2 was present in all proximal tubules except those in the subcapsular area; however, by 14 days, CP-2 was detected in all proximal tubules. This comparison of Sertoli cells and proximal tubule cells indicates that CP-2 content is determined by the maturity of a cell and not by the age of the animal.

Germ cell-Sertoli cell interactions. Studies of cyclic protein-2 in the seminiferous tubule.

This review briefly describes the discovery and isolation of a novel Sertoli cell product, cyclic protein-2, (CP-2) and the generation of an antiserum against this protein. Using this antiserum, we demonstrated a stage-specific change in the synthesis of CP-2 by Sertoli cells within intact seminiferous tubules; synthesis is maximal at stages VI and VIIa,b of the cycle and minimal at stage XII. That CP-2 is a product of Sertoli cells was confirmed by immunohistochemical analysis. Comparison of CP-2 and transferrin synthesis by immature (17-day) and mature (75-day) Sertoli cells within intact seminiferous tubules has documented a significant increase in the synthesis of both proteins during testicular maturation. It was noteworthy, however, that the increase in CP-2 synthesis was much greater than the increase in transferrin synthesis. These data in conjunction with previous comparisons of the stage-specific changes in CP-2 and transferrin synthesis and secretion led to the hypothesis that the synthesis of these two proteins is regulated by different cellular interactions. Examination of cultured Sertoli cells obtained from mature rats demonstrated that transferrin synthesis and secretion were stimulated by hormones and vitamins, whereas CP-2 synthesis and secretion were not significantly affected by the same factors. Therefore, these data demonstrate that hormonal regulation of transferrin synthesis by Sertoli cells differs from hormonal regulation of CP-2 synthesis. Indeed, our data suggest that CP-2 synthesis is not directly regulated by hormones and vitamins. Finally, we demonstrated that when Sertoli cells are separated from germ cells and the Sertoli cells placed in culture, the age-dependent increase in CP-2 synthesis, noted with cultured tubules, is lost. In contrast, significantly more transferrin is synthesized by primary cultures of Sertoli cells obtained from old animals than from young animals. Taken together, all of these data indicate that the regulation of CP-2 synthesis and secretion by the Sertoli cell is unique and is primarily stimulated by paracrine signals or direct cell contact with the germ cells. Which of these mechanisms of cell-cell communication in the testis is important to regulation of CP-2 synthesis by Sertoli cells is unknown. Neither do we know which spermatogenic cell type provides this stimulus. These issues can now be addressed, however, because we have developed the protocols for isolating and culturing Sertoli cells from mature rat testes.