Quality control of the elongation step of protein synthesis by tmRNP.

Trans-translation is a quality-control process, activated upon premature termination of protein elongation, which recycles stalled ribosomes and degrades incomplete polypeptides. These functions are facilitated by transfer-messenger RNA (tmRNA, also called 10Sa RNA or SsrA RNA), a small stable RNA molecule encoded by the SsrA gene found in bacteria, chloroplasts and mitochondria. Most tmRNAs consist of a tRNA- and an mRNA-like domain connected by up to four pseudoknots. Comparative sequence analysis provided the first insight into tmRNA secondary and three-dimensional structure. Studies of the E. coli tmRNA in vitro and in vivo demonstrated that tmRNA functions as a ribonucleoprotein (RNP) complex with elongation factor Tu (EF-Tu), protein SmpB and ribosomal protein S1. The tRNA-like and mRNA-like activities of tmRNA mark prematurely terminated proteins for degradation by attaching to their C-termini peptide tags, which are recognized by numerous proteases. Studies aimed at understanding the details of the molecular mechanisms of trans-translation are ongoing.

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.

Three-dimensional folding of the tRNA-like domain of Escherichia coli tmRNA.

UV irradiation of Escherichia coli tmRNA both on and off the ribosome induced covalent cross-links between its 3'- and its 5'-terminal segments. Cross-linking was unaffected in a molecule that lacked the tag-peptide codon region and pseudoknots 2, 3, and 4. Intact and truncated cross-linked tmRNAs were aminoacylated as efficiently as the respective nonirradiated molecules, suggesting that the added UV-induced bonds did not disturb tmRNA conformation. Using RNase H digestion followed by primer extension with reverse transcriptase, two cross-linked sites were identified within the tRNA-like region of tmRNA. The first was formed between nucleotides U9/U10 near the 5' end and nucleotides C346/U347 in the T loop. The second cross-link involved residues at positions 25-28 and 326-329 within helix 2a. Together with comparative sequence analysis, these findings yielded a three-dimensional model of the tRNA-like domain of E. coli tmRNA. Despite significant reduction of the D domain and the proximity of U9/U10 and C346/U347, the model closely resembles the L-shaped structure of canonical tRNA.

Expression and molecular characterization of estrogen receptor alpha messenger RNA in male reproductive organs of adult goats.

The fact that male estrogen receptor alpha (ERalpha) knockout mice are infertile indicates a role for this receptor in male reproduction. Here, objectives were to evaluate ERalpha expression in male goat reproductive tissues at the transcriptional level using RNase protection assay (RPA) and in situ hybridization (ISH), and to clone a partial cDNA for caprine ERalpha using reverse transcription-polymerase chain reaction (RT-PCR). For RPA and ISH procedures, a radiolabeled antisense cRNA probe, generated in vitro from the ovine oER8 cDNA template, was employed. Evaluations were made on individual samples obtained from adult goats. Labeled cRNA sense probe was used as a negative control in ISH. A 530-base pair amplicon was generated by RT-PCR from efferent ductules (EDs), epididymis (EP), and testis, cloned from the ED and EP, and sequenced. The caprine ERalpha (cERalpha) cDNA displayed 81%-96% sequence identity with that of other species. A signal indicative of ERalpha mRNA was identified by both RT-PCR and RPA in all tissues, but was strongest in the ED. Compared with ED, ERalpha signal was sixfold lower in the EP, and 66-fold lower in the testis. Similarly, strong ERalpha expression was observed in ED epithelium, whereas little or no signal was detected in EP or testis by ISH. Thus, among different segments of the male reproductive tract and testis, the highest level of ERalpha mRNA expression was found in epithelium of the ED.

tmRDB (tmRNA database).

The tmRNA database (tmRDB) is maintained at the University of Texas Health Science Center at Tyler, Texas, and accessible on the World Wide Web at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.++ +html. Mirror sites are located at Auburn University, Auburn, Alabama (http://www.ag.auburn.edu/mirror/tmRDB/) and the Institute of Biological Sciences, Aarhus, Denmark (http://www.bioinf.au. dk/tmRDB/). The tmRDB provides information and citation links about tmRNA, a molecule that combines functions of tRNA and mRNA in trans-translation. tmRNA is likely to be present in all bacteria and has been found in algae chloroplasts, the cyanelle of Cyanophora paradoxa and the mitochondrion of the flagellate Reclinomonas americana. This release adds 26 new sequences and corresponding predicted tmRNA-encoded tag peptides for a total of 86 tmRNAs, ordered alphabetically and phylogenetically. Secondary structures and three-dimensional models in PDB format for representative molecules are being made available. tmRNA alignments prove individual base pairs and are generated manually assisted by computational tools. The alignments with their corresponding structural annotation can be obtained in various formats, including a new column format designed to improve and simplify computational usability of the data.

Binding and cross-linking of tmRNA to ribosomal protein S1, on and off the Escherichia coli ribosome.

UV irradiation of an in vitro translation mixture induced cross-linking of 4-thioU-substituted tmRNA to Escherichia coli ribosomes by forming covalent complexes with ribosomal protein S1 and 16S rRNA. In the absence of S1, tmRNA was unable to bind and label ribosomal components. Mobility assays on native gels demonstrated that protein S1 bound to tmRNA with an apparent binding constant of 1 x 10(8) M(-1). A mutant tmRNA, lacking the tag coding region and pseudoknots pk2, pk3 and pk4, did not compete with full-length tmRNA, indicating that this region is required for S1 binding. This was confirmed by identification of eight cross-linked nucleotides: U85, located before the resume codon of tmRNA; U105, in the mRNA portion of tmRNA; U172 in pK2; U198, U212, U230 and U240 in pk3; and U246, in the junction between pk3 and pk4. We concluded that ribosomal protein S1, in concert with the previously identified elongation factor EF-Tu and protein SmpB, plays an important role in tmRNA-mediated trans-translation by facilitating the binding of tmRNA to ribosomes and forming complexes with free tmRNA.

Transit of tRNA through the Escherichia coli ribosome. Cross-linking of the 3' end of tRNA to specific nucleotides of the 23 S ribosomal RNA at the A, P, and E sites.

When bound to Escherichia coli ribosomes and irradiated with near-UV light, various derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at the 3' terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner. A and P site-bound tRNAs, whose 3' termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33. The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U2506 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer. In particular, the 3'-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNA(Phe), remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site. Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction. We show that the 3'-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center. In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm.

Kinetic parameters for tmRNA binding to alanyl-tRNA synthetase and elongation factor Tu from Escherichia coli.

Aminoacylation and transportation of tmRNA to stalled ribosomes constitute prerequisite steps for trans-translation, a process facilitating the release of stalled ribosomes from 3' ends of truncated mRNAs and the degradation of incompletely synthesized proteins. Kinetic analysis of the aminoacylation of tmRNA indicates that tmRNA has both a lower affinity and a lower turnover number than cognate tRNA(Ala) for alanyl-tRNA synthetase, resulting in a 75-fold lower k(cat)/K(M) value. The association rate constant of Ala-tmRNA for elongation factor Tu in complex with GTP is about 150-fold lower than that of Ala-tRNA(Ala), whereas its dissocation rate constant is about 5-fold lower. These observations can be interpreted to suggest that additional factors facilitate tmRNA binding to ribosomes.

tmRDB (tmRNA database).

The tmRNA database (tmRDB) is maintained at the University of Texas Health Science Center at Tyler, Texas, and is accessible on the WWW at URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.++ +html. A tmRDB mirror site is located on the campus of Auburn University, Auburn, Alabama, reachable at the URL http://www.ag.auburn.edu/mirror/tmRDB/. Since April 1997, the tmRDB has provided sequences of tmRNA (previously called 10Sa RNA), a molecule present in most bacteria and some organelles. This release adds 17 new sequences for a total of 60 tmRNAs. Sequences and corresponding tmRNA-encoded tag peptides are tabulated in alphabetical and phylo-genetic order. The updated tmRNA alignment improves the secondary structures of known tmRNAs on the level of individual basepairs. tmRDB also provides an introduction to tmRNA function in trans-translation (with links to relevant literature), a limited number of tmRNA secondary structure diagrams, and numerous three-dimensional models generated interactively with the program ERNA-3D.

Comparative sequence analysis of tmRNA.

Minimal secondary structures of the bacterial and plastid tmRNAs were derived by comparative analyses of 50 aligned tmRNA sequences. The structures include 12 helices and four pseudoknots and are refinements of earlier versions, but include only those base pairs for which there is comparative evidence. Described are the conserved and variable features of the tmRNAs from a wide phylogenetic spectrum, the structural properties specific to the bacterial subgroups and preliminary 3-dimensional models from the pseudoknotted regions.

The tmRNA database (tmRDB).

As of September, 1998, a total of 43 sequences are contained within the tmRNA database (tmRDB). The tmRNA sequences are arranged alphabetically and ordered phylogenetically. The alignment of the tmRNAs emphasizes the basepairs that are supported by comparative sequence analysis and establishes minimal secondary structures for the known tmRNAs. A corresponding alignment of the predicted tmRNA-encoded tag peptides is presented. The tmRDB also offers a small number of RNA secondary structure diagrams and PDB-formatted three-dimensional models generated with the program ERNA-3D. The data are available freely at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.++ +html

Ribosomal protein L27 participates in both 50 S subunit assembly and the peptidyl transferase reaction.

Protein L27 has been implicated as a constituent of the peptidyl transferase center of the Escherichia coli 50 S ribosomal subunit by a variety of experimental observations. To define better the functional role of this protein, we constructed a strain in which the rpmA gene, which encodes L27, was replaced by a kanamycin resistance marker. The deletion mutant grows five to six times slower than the wild-type parent and is both cold- and temperature-sensitive. This phenotype is reversed when L27 is expressed from a plasmid-borne copy of the rpmA gene. Analysis of ribosomes from the L27-lacking strain revealed deficiencies in both the assembly and activity of the 50 S ribosomal subunits. Although functional 50 S subunits are formed in the mutant, an assembly "bottleneck" was evidenced by the accumulation of a prominent 40 S precursor to the 50 S subunit which was deficient in proteins L16, L20, and L21, as well as L27. In addition, the peptidyl transferase activity of 70 S ribosomes containing mutant 50 S subunits was determined to be three to four times lower than for wild-type ribosomes. Ribosomes lacking L27 were found to be impaired in the enzymatic binding of Phe-tRNAPhe to the A site, although the interaction of N-acetyl-Phe-tRNAPhe with the P site was largely unperturbed. We therefore infer that L27 contributes to peptide bond formation by facilitating the proper placement of the acceptor end of the A-site tRNA at the peptidyl transferase center.

The tmRNA database (tmRDB).

This first release of the tmRNA database (tmRDB) contains 19 tmRNA sequences, a tmRNA sequence alignment with emphasis of base pairs that are supported by comparative sequence analysis, and a tabulation of tmRNA-encoded tag peptides. The tmRNADB also offers an RNA secondary structure diagram of the Escherichia coli tmRNA, as well as PDB-formatted coordinates for three-dimensional modeling. The data are available on the World Wide Web at http://www.uthct. edu/tmRDB/tmRDB.html

Peptidyl transferase and beyond.

The peptidyl transferase center of the Escherichia coli ribosome encompasses a number of 50S-subunit proteins as well as several specific segments of the 23S rRNA. Although our knowledge of the role that both ribosomal proteins and 23S rRNA play in peptide bond formation has steadily increased, the location, organization, and molecular structure of the peptidyl transferase center remain poorly defined. Over the past 10 years, we have developed a variety of photoaffinity reagents and strategies for investigating the topography of tRNA binding sites on the ribosome. In particular, we have used the photoreactive tRNA probes to delineate ribosomal components in proximity to the 3' end of tRNA at the A, P, and E sites. In this article, we describe recent experiments from our laboratory which focus on the identification of segments of the 23S rRNA at or near the peptidyl transferase center and on the functional role of L27, the 50S-subunit protein most frequently labeled from the acceptor end of A- and P-site tRNAs. In addition, we discuss how these results contribute to a better understanding of the structure, organization, and function of the peptidyl transferase center.

Synthesis of 2,6-diazido-9-(beta-D-ribofuranosyl)purine 3',5'-bisphosphate: incorporation into transfer RNA and photochemical labeling of Escherichia coli ribosomes.

2,6-Diazido-9-(beta-D-ribofuranosyl)purine was prepared by the reaction of 2,6-dichloro-9-(beta-D-ribofuranosyl)purine with sodium azide. The nucleoside was bisphosphorylated with pyrophosphoryl chloride to form 2,6-diazido-9-(beta-D-ribofuranosyl)purine 3',5'-bisphosphate. This product was labeled with 32P using T4 polynucleotide kinase to exchange the 5' phosphate with the gamma phosphate of [gamma-32P]ATP. When yeast tRNA(Phe) containing 2,6-diazido-9-(beta-D-ribofuranosyl)purine at the 3' terminus was bound to the P site of the Escherichia coli ribosome in the presence of poly(U) and irradiated with 300-nm light, the photoreactive tRNA derivative became cross-linked exclusively to the 50S subunit. The label was attached to proteins L27 and L33 as well as to the 23S rRNA.

Recombinant photoreactive tRNA molecules as probes for cross-linking studies.

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.

Mapping the central fold of tRNA2(fMet) in the P site of the Escherichia coli ribosome.

4-Thiouridine (s4U), a photoreactive analog of uridine, was randomly incorporated into tRNA2(fMet) precursor molecules by transcription with T7 RNA polymerase. The s4U-containing transcripts were trimmed at their 5'-ends with RNase P RNA to yield mature tRNA2(fMet). The photoreactive tRNA2(fMet) derivatives were aminoacylated and bound to the P site of 70S ribosomes from Escherichia coli in the presence of a poly(A,G,U) template. Irradiation of the complexes at 300 nm resulted in the covalent cross-linking of tRNA2(fMet) to ribosomal proteins and rRNAs within both the 50S and 30S subunits. The labeled proteins were identified as L1, L27, and S19. 50S-subunit proteins L1 and L27 were attached to nucleotide U17 or U17.1 within the D loop of tRNA2(fMet), whereas 30S-subunit protein S19 was cross-linked to nucleotide U47 in the variable loop. Both of these sites occur in or near the central fold of the tRNA. These results permit us to map the D loop of P site-bound tRNA to the region between the central protuberance and the L1 ridge on the 50S ribosomal subunit, while the variable loop can be placed above the cleft on the head of the 30S subunit.

Topography of the E site on the Escherichia coli ribosome.

Three photoreactive tRNA probes have been utilized in order to identify ribosomal components that are in contact with the aminoacyl acceptor end and the anticodon loop of tRNA bound to the E site of Escherichia coli ribosomes. Two of the probes were derivatives of E. coli tRNA(Phe) in which adenosines at positions 73 and 76 were replaced by 2-azidoadenosine. The third probe was derived from yeast tRNA(Phe) by substituting wyosine at position 37 with 2-azidoadenosine. Despite the modifications, all of the photoreactive tRNA species were able to bind to the E site of E. coli ribosomes programmed with poly(A) and, upon irradiation, formed covalent adducts with the ribosomal subunits. The tRNA(Phe) probes modified at or near the 3' terminus exclusively labeled protein L33 in the 50S subunit. The tRNA(Phe) derivative containing 2-azidoadenosine within the anticodon loop became cross-linked to protein S11 as well as to a segment of the 16S rRNA encompassing the 3'-terminal 30 nucleotides. We have located the two extremities of the E site-bound tRNA on the ribosomal subunits according to the positions of L33, S11 and the 3' end of 16S rRNA defined by immune electron microscopy. Our results demonstrate conclusively that the E site is topographically distinct from either the P site or the A site, and that it is located alongside the P site as expected for the tRNA exit site.

Photochemical cross-linking of the anticodon loop of yeast tRNA(Phe) to 30S-subunit protein S7 at the ribosomal A and P sites.

Yeast tRNA(Phe), containing the photoreactive nucleoside 2-azidoadenosine at position 37 within the anticodon loop, has been cross-linked to the aminoacyl-tRNA (A) and peptidyl-tRNA (P) binding sites of the Escherichia coli ribosome. The 30S subunit was exclusively labeled in each case, and cross-linking occurred to both protein and 16S rRNA. Electrophoretic and immunological analyses demonstrated that S7 was the only 30S-subunit protein covalently attached to the tRNA. However, digestion of the A and P site-labeled S7 with trypsin revealed a unique pattern of cross-linked peptide(s) at each site. Thus, while the anticodon loop of tRNA is in close proximity to protein S7 at both the A and P sites, it neighbors a different portion of the protein molecule in each. The placement of the aminoacyl- and peptidyl-tRNA binding sites is discussed in relationship to recent models of the 30S ribosomal subunit.