Contributions of pseudoknots and protein SmpB to the structure and function of tmRNA in trans-translation

Bacteria contain transfer-messenger RNA (tmRNA), a molecule that during trans-translation tags incompletely translated proteins with a small peptide to signal the proteolytic destruction of defective polypeptides. TmRNA is composed of tRNA- and mRNA-like domains connected by several pseudoknots. Using truncated ribosomal protein L27 as a reporter for tagging in vitro and in vivo, we have developed exceptionally sensitive assays to study the role of Escherichia coli tmRNA in trans-translation. Site-directed mutagenesis experiments showed that pseudoknot 2 and the abutting helix 5 were particularly important for the binding of ribosomal protein S1 to tmRNA. Pseudoknot 4 not only facilitated tmRNA maturation but also promoted tagging. In addition, the three pseudoknots (pk2 to pk4) were shown to play a significant role in the proper folding of the tRNA-like domain. Protein SmpB enhanced tmRNA processing, suggesting a new role for SmpB in trans-translation. Taken together, these results provide unanticipated insights into the functions of the pseudoknots and protein SmpB during tmRNA folding, maturation, and protein synthesis.     

Interaction analysis between tmRNA and SmpB from Thermus thermophilus

Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.     

Cell cycle-regulated degradation of tmRNA is controlled by RNase R and SmpB

The production and removal of regulatory RNAs must be controlled to ensure proper physiological responses. SsrA RNA (tmRNA), a regulatory RNA conserved in all bacteria, is cell cycle regulated and is important for control of cell cycle progression in Caulobacter crescentus. We report that RNase R, a highly conserved 3' to 5' exoribonuclease, is required for the selective degradation of SsrA RNA in stalked cells. Purified RNase R degrades SsrA RNA in vitro, and is kinetically competent to account for all SsrA RNA turnover. SmpB, a tmRNA-binding protein, protects SsrA RNA from RNase R degradation in vitro, and the levels of SmpB protein during the cell cycle correlate with SsrA RNA stability. These results suggest that SmpB binding controls the timing of SsrA RNA degradation by RNase R. We propose a model for the regulated degradation of SsrA RNA in which RNase R degrades SsrA RNA from a non-tRNA-like 3' end, and SmpB specifically protects SsrA RNA from RNase R. This model explains the regulation of SsrA RNA in other bacteria, and suggests that a highly conserved regulatory mechanism controls SsrA activity.     

A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif

A 64 kd protein was shown to bind to RNAs that contain functional polyadenylation signals by a UV cross-linking procedure in which label was transferred from RNA substrate to protein in cell-free polyadenylation extracts. The 64 kd nuclear protein bound specifically to three different substrates (adenovirus type 5 L3, SV40 early, and SV40 late polyadenylation domains), as determined by competition experiments and partial protease analysis. Deleted derivatives of the SV40 late substrate that retained the sequence 5'-CUGCAAUAAACAAGUU-3' were able to bind the 64 kd polypeptide. This sequence contains the canonical AAUAAA element that has been shown to be indispensable for polyadenylation. A single nucleotide change, converting AAUAAA to AAGAAA, prevented binding of the 64 kd moiety. The 64 kd protein was shown to be distinct from poly(A) polymerase by biochemical fractionation.     

ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences

A computer program, ARAGORN, identifies tRNA and tmRNA genes. The program employs heuristic algorithms to predict tRNA secondary structure, based on homology with recognized tRNA consensus sequences and ability to form a base-paired cloverleaf. tmRNA genes are identified using a modified version of the BRUCE program. ARAGORN achieves a detection sensitivity of 99% from a set of 1290 eubacterial, eukaryotic and archaeal tRNA genes and detects all complete tmRNA sequences in the tmRNA database, improving on the performance of the BRUCE program. Recently discovered tmRNA genes in the chloroplasts of two species from the ‘green’ algae lineage are detected. The output of the program reports the proposed tRNA secondary structure and, for tmRNA genes, the secondary structure of the tRNA domain, the tmRNA gene sequence, the tag peptide and a list of organisms with matching tmRNA peptide tags.     

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.     

A metalloantibody that irreversibly binds a protein antigen

Antibody affinity is critically important in therapeutic applications, as well as steady state diagnostic assays. Picomolar affinity antibodies, approaching the association limit of protein-protein interactions, have been discovered for highly potent antigens, but even such high-affinity binders have off-rates sufficient to negate therapeutic efficacy. To cross this affinity threshold, antibodies that tether their targets in a manner other than reversible non-covalent interaction will be required. Here we report the design and construction of an antibody that forms an irreversible complex with a protein antigen in a metal-dependent reaction. The complex resists thermal and chemical denaturation, as well as attempts to remove the coordinating metal ion. Such irreversibly binding antibodies could facilitate the development of next generation "reactive antibody" therapeutics and diagnostics.     

The role of SmpB and the ribosomal decoding center in licensing tmRNA entry into stalled ribosomes

In bacteria, stalled ribosomes are recycled by a hybrid transfer-messenger RNA (tmRNA). Like tRNA, tmRNA is aminoacylated with alanine and is delivered to the ribosome by EF-Tu, where it reacts with the growing polypeptide chain. tmRNA entry into stalled ribosomes poses a challenge to our understanding of ribosome function because it occurs in the absence of a codon-anticodon interaction. Instead, tmRNA entry is licensed by the binding of its protein partner, SmpB, to the ribosomal decoding center. We analyzed a series of SmpB mutants and found that its C-terminal tail is essential for tmRNA accommodation but not for EF-Tu activation. We obtained evidence that the tail likely functions as a helix on the ribosome to promote accommodation and identified key residues in the tail essential for this step. In addition, our mutational analysis points to a role for the conserved K(131)GKK tail residues in trans-translation after peptidyl transfer to tmRNA, presumably EF-G-mediated translocation or translation of the tmRNA template. Surprisingly, analysis of A1492, A1493, and G530 mutants reveals that while these ribosomal nucleotides are essential for normal tRNA selection, they play little to no role in peptidyl transfer to tmRNA. These studies clarify how SmpB interacts with the ribosomal decoding center to license tmRNA entry into stalled ribosomes.     

EVI5 is a novel centrosomal protein that binds to alpha- and gamma-tubulin

The human EVI5 protein carries a TBC domain indicative of Rab GTPase activating protein (GAP) activity, and an extensive coiled-coil motif in the C-terminal region. EVI5 is ubiquitously expressed in adult, fetal, and cancer tissues and exists as two mRNA species resulting from differential use of polyadenylation signals. Western blot analysis suggests that different molecular weight protein species are probably generated by posttranslational modification. FPLC analysis demonstrates that EVI5 protein can form dimers and confocal microscopy indicates that EVI5, in addition to a diffuse localization in the nucleus, also preferentially localizes to the pericentriolar material in interphase cells. Immunoprecipitation and GST pull-down experiments demonstrate that EVI5 exists in complexes with both alpha- and gamma-tubulin. Both interactions are localized to the N-terminal part of the EVI5 protein. Thus, EVI5 is a novel centrosomal protein with a complex expression pattern and subcellular localization, possibly involved in centrosome stability and dynamics.     

LaRbp38: a Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.     

Characterization of a cDNA encoding a novel heat-shock protein that binds to calmodulin.

A cDNA clone (pTCB48) encoding a calmodulin-binding protein was isolated by screening a lambda ZAPII cDNA expression library constructed from cell cultures of heat-shocked tobacco (Nicotiana tabacum L. cv Wisconsin-38) with metabolically labeled [35S]calmodulin. Calmodulin gel overlay analysis indicated that pTCB48 generated major peptides of 53, 36, and 22 kD and two minor peptides of 37 and 16 kD that bound calmodulin in a Ca(2+)-dependent manner. Deletion analysis of pTCB48 indicated that these and the minor calmodulin-binding proteins resulted from the insert. A probe made from the cDNA insert recognized two bands with sizes of 2.1 and 1.8 kb on northern blot analysis. Both species of RNAs were undetectable in the control and were induced after 15 min of heat-shock treatment at 38 degrees C. The intensity of the two bands reached maximum after 1.5 h of heat-shock treatment. The cDNA clone was not full length; however, the complete sequence was determined by 5' rapid amplification of cDNA ends using nested antisense primers. The full-length cDNA contains 1648 bp and a single open reading frame of 1347 bp and is expected to encode a protein of approximately 50 kD. No significant homology with other reported genes and proteins was found. Structural predictions, deletion analysis, and gel overlay analysis suggested that the calmodulin-binding domain was a basic amphiphilic alpha-helix near the C terminus of the protein. The strong induction of the mRNA for this protein suggests a role for Ca2+/calmodulin-mediated process in the heat-shock response.     

Hinderin,a five-domains protein including coiled-coil motifs that binds to SMC3

Background  

The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions.     

Isolation of a protein fraction that binds preferentially to chicken middle repetitive DNA

We have fractionated oviduct tissue extracts by using a combination of ion-exchange and DNA-Sephadex chromatography. By comparing the electrophoretic patterns of proteins eluted from competing specific and nonspecific DNA columns, we isolated a fraction which bound with specificity to columns containing the chicken middle repetitive sequence "CR1". This fraction showed a clear preference for binding to separate, cloned CR1 fragments derived from either the 5' or the 3' transition region of the ovalbumin gene domain when examined by using nitrocellulose filter binding assays. To localize the protein binding site, a CR1 clone was digested with various restriction enzymes, and the resulting fragments were examined for preferential protein binding. Results suggest that the binding site lies within a 39-nucleotide sequence which is highly conserved among different CR1 elements. This finding represents the first isolation of a protein which demonstrates a preference for binding to a middle repetitive sequence and suggests that this interaction may have a biological role. The DNA column competition adsorption method should have general application to the isolation of other gene-regulating proteins possessing DNA sequence preference.     

The role of SmpB protein in trans-translation

The function of SmpB protein in the trans-translation system was evaluated using the well-defined cell-free translation system consisting of purified ribosome, alanyl-tRNA synthetase and elongation factors. The analysis showed that SmpB protein enhances alanine-accepting activity of tmRNA and that SmpB protein and tmRNA are sufficient to complete the trans-translation process in the presence of translational components. Moreover, SmpB is indispensable in the addition of tag-peptide onto ribosomes by tmRNA. In particular, the A-site binding of tmRNA is inhibited in the absence of SmpB.     

Towards a three-alpha-helix bundle protein that binds volatile general anesthetics

The general anesthetics halothane and chloroform are capable of binding to synthetic water-soluble four-alpha-helix bundles, which model the putative in vivo receptors. In this study, we investigate the binding of these anesthetics to synthetic water-soluble three-alpha-helix bundles. A series of variants containing up to four X-to-Ala and up to four X-to-Met substitutions was made; and the effect of these substitutions on structure, stability and anesthetic binding affinity was examined. Generally, the amount of alpha-helix and the stability of the three-alpha-helix bundles decreased as the number of X-to-Ala substitutions increased. A concomitant red-shift in tryptophan fluorescence lambdamax was seen, suggesting an increased flexibility of the native structure. Up to four X-to-Met substitutions had little effect on the amount of alpha-helix, but an increase in tryptophan lambdamax was seen for the variants with three and four methionine substitutions. The exceptions were a) a variant with a clustering of alanine and methionine residues at one end of the three-alpha-helix bundle, suggesting a gate structure that can admit ligand molecules; and b) a variant with a single Leu35Ala substitution, suggesting that at select positions, the size of the side chain is important for defining anesthetic binding affinity.