tRNA Sec Research Articles

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA.

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.

Inhibition of Selenoprotein Synthesis by Selenocysteine tRNA[Ser]Sec Lacking Isopentenyladenosine

A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A. To address the role of the modified nucleoside in this tRNA, we constructed a site-specific mutation, which eliminates the site of isopentenylation, in the Xenopus tRNA([Ser]Sec) gene. Transfection of the mutant tRNA([Ser]Sec) gene resulted in 80% and 95% reduction in the expression of co-transfected selenoprotein genes encoding type I and II iodothyronine deiodinases, respectively. A similar decrease in type I deiodinase synthesis was observed when transfected cells were treated with lovastatin, an inhibitor of the biosynthesis of the isopentenyl moiety. Neither co-transfection with the mutant tRNA gene nor lovastatin treatment reduced type I deiodinase mRNA levels. Also, mutant tRNA expression did not alter initiation of translation or degradation of the type I deiodinase protein. Furthermore, isopentenylation of tRNA([Ser]Sec) was not required for synthesis of Sec on the tRNA. We conclude that isopentenylation of tRNA([Ser]Sec) is required for efficient translational decoding of UGA and synthesis of selenoproteins.

Methylation of the ribosyl moiety at position 34 of selenocysteine tRNA[Ser]Sec is governed by both primary and tertiary structure.

The selenocysteine (Sec) tRNA[Ser]Sec population in higher vertebrates consists of two major isoacceptors that differ from each other by a single nucleoside modification in the wobble position of the anticodon (position 34). One isoacceptor contains 5-methylcarboxymethyluridine (mcmU) in this position, whereas the other contains 5-methylcarboxymethyluridine-2'-O-methylribose (mcmUm). The other modifications in these tRNAs are N6-isopentenyladenosine (i6A), pseudouridine (psi), and 1-methyladenosine (m1A) at positions 37, 55, and 58, respectively. As methylation of the ribose at position 34 is influenced by the intracellular selenium status and the presence of this methyl group dramatically alters tertiary structure, we investigated the effect of the modifications at other positions as well as tertiary structure on its formation. Mutations were introduced within a synthetic gene encoded in an expression vector, transcripts generated and microinjected into Xenopus oocytes, and the resulting tRNA products analyzed for the presence of modified bases. The results suggest that efficient methylation of mcmU to yield mcmUm requires the prior formation of each modified base and an intact tertiary structure, whereas formation of modified bases at other positions, including mcmU, is not as stringently connected to precise primary and tertiary structure. These results, along with the observations that methylation of mcmU is enhanced in the presence of selenium and that this methyl group affects tertiary structure, further suggest that the mcmUm isoacceptor must have a role in selenoprotein synthesis different from that of the mcmU isoacceptor.

Eukaryotic selenocysteine tRNA has the 9/4 secondary structure

There are two secondary structure models for the eukaryotic selenocysteine (Sec) tRNA Sec. One model, the 9/4 structure, was experimentally tested and possesses acceptor and T-stems with 9 and 4 bp, respectively [Sturchler et al., 1993; Hubert et al., 1998]. The other one, the 7/5 secondary structure with a bulge in the T-stem, was derived from theoretical calculation [Ioudovitch and Steinberg, 1999]. In this report, we show more experimental results supporting the 9/4 secondary structure. Several tRNA Sec mutants, whose secondary structure can adopt only the 9/4 structure, were active for serylation and selenylation. Some mutants that cannot base-pair between positions 26 and 44 to provide the 6 bp anticodon stem were still active, inconsistent with the model by Steinberg. We also show that the orientation of the V-arm directly or indirectly influences the selenylation activity, and that the rigid 6 bp D-stem is important. Finally, we conclude that all tRNAs Sec possess the 13 bp domain II made by the stacking of the colinear AA and T-stems, whether they present the 9/4 structure in Eukarya and Archaea or the 8/5 structure in bacteria.

Selenium Metabolism in Drosophila: CHARACTERIZATION OF THE SELENOCYSTEINE tRNA POPULATION

The selenocysteine (Sec) tRNA population in Drosophila melanogaster is aminoacylated with serine, forms selenocysteyl-tRNA, and decodes UGA. The Km of Sec tRNA and serine tRNA for seryl-tRNA synthetase is 6.67 and 9.45 nM, respectively. Two major bands of Sec tRNA were resolved by gel electrophoresis. Both tRNAs were sequenced, and their primary structures were indistinguishable and colinear with that of the corresponding single copy gene. They are 90 nucleotides in length and contain three modified nucleosides, 5-methylcarboxymethyluridine, N6-isopentenyladenosine, and pseudouridine, at positions 34, 37, and 55, respectively. Neither form contains 1-methyladenosine at position 58 or 5-methylcarboxymethyl-2'-O-methyluridine, which are characteristically found in Sec tRNA of higher animals. We conclude that the primary structures of the two bands of Sec tRNA resolved by electrophoresis are indistinguishable by the techniques employed and that Sec tRNAs in Drosophila may exist in different conformational forms. The Sec tRNA gene maps to a single locus on chromosome 2 at position 47E or F. To our knowledge, Drosophila is the lowest eukaryote in which the Sec tRNA population has been characterized to date.

A G·U base pair in the eukaryotic selenocysteine tRNA is important for interaction with SePF, the putative selenocysteine-specific elongation factor

In Escherichia coli, selenocysteine biosynthesis and incorporation into selenoproteins requires the action of four gene products, including the specialized selenocysteine tRNA Sec and elongation factor SELB, different from the universal EF-Tu. In this regard, the situation is less clear in eukaryotes, but we previously reported the existence of SePF, a putative SELB homologue. The secondary structure of the tRNA Sec differs slightly in eukaryotes, due to a change in the lengths of several stems. Two non-Watson-Crick base pairs, G5a·U67b and U6·U67, reside in the acceptor stem and are conserved in the course of evolution. Since it has already been reported that changing them to Watson-Crick base pairs did not affect the serylation or selenylation levels of tRNA Sec, we asked whether these non-Watson-Crick base pairs are required for the interaction with SePF. To this end, tRNA Sec variants carrying Watson-Crick changes at these positions were tested for their ability to maintain the interaction with SePF. In these assays, the tRNA Sec-SePF interaction was determined by the protective action it confers against hydrolysis of the amino acid ester bond, under basic conditions. All the changes introduced at U6·U67 did not significantly affect the interaction. Interestingly, however, the G5a·U67b to G5a-C67b substitution was sufficient, by itself, to lead to unprotection of the ester bond. Therefore, our finding strongly suggests that SePF is unable to interact with a tRNA Sec mutant version carrying a Watson-Crick G5a-C67b instead of the wild-type G5a·U67b base pair, establishing that G5a·U67b constitutes a structural determinant for SePF interaction.

Minimal tRNA Ser and tRNA Sec substrates for human seryl-tRNA synthetase: contribution of tRNA domains to serylation and tertiary structure

The recognition process of tRNA Ser and tRNA Sec by human seryl-tRNA synthetase (SerRS) was studied using T7 transcripts representing defined regions of human tRNA Ser or tRNA Sec and the influence of the tRNA elements on serylation and tertiary structure was elucidated. The anticodon arms of both tRNAs showed no contribution to serylation in contrast to the acceptor stems and the long extra arms. D and T arms were only involved in formation of the L-shaped tRNA structure, not in the recognition process between tRNAs and SerRS. This is the first report of microhelices adapted from human tRNAs being aminoacylated by their homologous synthetase.

The dual identities of mammalian tRNA(Sec) for SerRS and selenocysteine synthase.

Se is an essential trace element and is found as a selenocysteine in the active site of Se-enzymes, such as glutathione peroxidase. tRNASec is first aminoacylated with serine by Ser RS and further is converted to selenocysteyl-tRNA by selenocysteine synthase. Mammalian selenocysteine tRNA has dual identities with Ser RS and selenocysteine synthase. Key identity elements for selenocysteine synthase are the long 9 bp AA- and long 6 bp D-stems. Major serine tRNA was converted to a mutant with a 9 bp AA-stem and 6 bp D-stem, instead of a 7 bp AA-stem and 3 bp D-stem. This mutant was active for selenylation as well as serylation. The relative kinetic parameter (Vmax/Km) of the mutant was 0.052 of the value (1.00) of wild-type Sec tRNA. This low value suggests that there is an unknown fine base specific for selenocysteine synthase. For serylation, mutant having 12 bp and wild type tRNASec having 13 bp of the total length of AA- + T-stems were active but the mutants having 11 or 14 bp were inactive. This shows that SerRS measures the distance between the discrimination base and long extra arm for recognition of tRNASer.

Replenishment of selenium deficient rats with selenium results in redistribution of the selenocysteine tRNA population in a tissue specific manner

We reported previously that the selenium status of rats influences both the steady-state levels and distributions of two selenocysteine tRNA isoacceptors and that these isoacceptors differ by a single methyl group attached to the ribosyl moiety at position 34. In this study, we demonstrate that repletion of selenium-deficient rats results in a gradual, tissue-dependent shift in the distribution of these isoacceptors. Rats fed a selenium-deficient diet possess a greater abundance of the species unmethylated on the ribosyl moiety at position 34 compared to the form methylated at this position. A redistribution of the Sec–tRNA isoacceptors occurred in tissues of selenium-supplemented rats whereby the unmethylated form gradually shifted toward the methylated form. This was true in each of four tissues examined, muscle, kidney, liver and heart, although the rate of redistribution was tissue-specific. Muscle manifested a predominance of two minor serine isoacceptors under conditions of extreme selenium-deficiency which also appeared to respond to selenium. Ribosomal binding studies revealed that one of the two additional isoacceptors decodes the serine codeword, AGU, and the second decodes the serine codeword, UCU. Interestingly, muscle and heart were the slower tissues to return to a `selenium adequate' tRNA distribution pattern.

Selenocysteine tRNA[Ser]Sec levels and selenium-dependent glutathione peroxidase activity in mouse embryonic stem cells heterozygous for a targeted mutation in the tRNA[Ser]Sec gene.

To investigate the effect of a reduced level of selenocysteine (Sec) tRNA[Ser]Sec in selenoprotein biosynthesis, two mouse embryonic stem (ES) cell lines heterozygous for the corresponding gene were generated by homologous recombination of the host genome with targeting vectors encoding a deleted or a disrupted tRNA[Ser]Sec gene. The presence of a single functional gene in ES cells afforded us an opportunity to determine directly in the cell line the effect of reduced gene dosage on (1) the levels of the Sec tRNA[Ser]Sec population, (2) the distributions of the isoacceptors within the Sec tRNA population, and (3) selenoprotein biosynthesis. We therefore determined the amounts and distributions of the two major tRNA[Ser]Sec isoacceptors, designated mcm5U and mcm5Um, within the Sec tRNA population and determined the activity of the anti-oxidant, selenium-containing glutathione peroxidase (GPx) in the heterozygotes and in wild type cells grown in media with and without added selenium. The level of the Sec tRNA[Ser]Sec population in the heterozygotes was approximately 60% of that of wild type cells grown in media under normal conditions, while the ratio of the mcmU isoacceptor in wild type vs mutant cells was approximately 2:1 and of the mcmUm isoacceptor approximately 1:1. In the presence of media supplemented with selenium, the Sec tRNA[Ser]Sec population increased about 20% in wild type cells and virtually not all in heterozygous cells, and the level of the Sec tRNA[Ser]Sec population was, therefore, approximately 50% of that of wild type cells. GPx activity was indistinguishable among these cell lines in either selenium-supplemented or unsupplemented media, indicating that the resultant changes in tRNA[Ser]Sec levels did not have a measurable effect on GPx biosynthesis.

Selenocysteine Synthesis in Mammalia: An Identity Switch From tRNA Serto tRNA Sec

The mechanism of selenocysteine insertion into proteins is distinct from all other amino acids in all lines of descent in that it needs specific protein cofactors and a structurally unique tRNA Sec. It is first aminoacylated with serine and further recognized among all other serylated serine isoacceptors by a selenocysteine synthase and is converted to selenocysteyl-tRNA SecWe present here the complete set of identity elements for selenylation of mammalian seryl-tRNA Secand show that the transplantation of these elements into normal serine tRNA allows its selenylation. Four particular structural motifs differentiate eukaryotic tRNA Secfrom normal tRNA Ser: the orientation of the extra arm, the short 4 bp TΨC-stem, the extra long 9 bp acceptor-stem and the elongated 6 bp dihydrouridine-stem. Only the last two are essential and only together sufficient for selenocysteine synthesis, whereby the additional base-pairs of the acceptor-stem may be replaced by non-paired nucleotides. Each exchange of the first three structural motifs mentioned above between tRNA Serand tRNA Secresulted in a significant loss of serylation, indicating that the overall composition of particular structure elements is necessary to maintain normal functions of tRNA Sec. Since we find that all seryl-tRNAs which are selenylated are also substrates for serine phosphorylation we propose that phosphoseryl- tRNA Secis a storage form of seryl-tRNA Sec.

Analysis of the Selenocysteine tRNA[SER]SECGene Transcriptionin VitroUsingXenopusOocyte Extracts

Anin vitrotranscription system was devised using aXenopusoocyte S150 extract for analyzing expression of the Sec tRNA[Ser]Secgene. The activator element, located at about −200 that is required for maximal expression of the tRNA[Ser]Secgenein vivo,had no effect on tRNA[Ser]Sectranscriptionin vitro. In vitrotranscription of this gene even tolerated mutations within or deletion of the PSE, showing marked contrast with the requirements observedin vivo.However, the TATA box was indispensable for basal level expression of the gene bothin vivoandin vitro.The region spanning from the TATA box to the transcription start point (−33 to −1) was sufficient for Pol III to recognize the tRNA[Ser]Secgene promoterin vitro.

Antideterminants present in minihelix(Sec) hinder its recognition by prokaryotic elongation factor Tu.

During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNA Sec form ternary complexes with activated elongation factor. tRNA Sec is bound by its own translation factor, an elongation factor analogue, e.g. the SELB factor in prokaryotes. An apparent reason for this discrimination could be related to the unusual length of tRNA Sec amino acid-acceptor branch formed by 13 bp. However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNA Asp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNA Sec prevent its recognition by EF-Tu-GTP. A stepwise mutational analysis of a minihelix derived from tRNA Sec in which sequence elements of tRNA Asp were introduced showed that the sequence of the amino acid- acceptor branch of Escherichia coli tRNA Sec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP. This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA Sec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem. The function of this C7.G66/G49.U65/C50.G64 box was tested by its transplantation into a minihelix derived from tRNA Asp, abolishing its recognition by EF-Tu-GTP. The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.

RNAs mediating cotranslational insertion of selenocysteine in eukaryotic selenoproteins

Selenocysteine, a selenium-containing analog of cysteine, is found in the prokaryotic and eukaryotic kingdoms in active sites of enzymes involved in oxidation-reduction reactions. Its biosynthesis and cotranslational insertion into selenoproteins is performed by an outstanding mechanism, implying the participation of several gene products. The tRNA Sec is one of these. In eukaryotes, its transcription mode by RNA polymerase III differs from that of classical tRNA genes, both at the level of the promoter elements and transcription factors involved. In addition, enhanced transcription is afforded by a newly characterized zinc finger activator. Not only transcription of the gene, but also the tRNA Sec itself is atypical since its 2D and 3D structures exhibit features which set it apart from classical tRNAs. Decoding of eukaryotic selenocysteine UGA codons requires a stem-loop structure in the 3′UTR of mRNAs, the selenocysteine insertion sequence (SECIS) element. Structure probing and sequence comparisons led us to propose a 2D structure model for the SECIS element, containing a novel RNA motif composed of four consecutive non-Watson-Crick base-pairs. A 3D model, rationalizing the accessibility data, was elaborated by computer modeling. It yields indicative or suggestive evidence for the role that could play some conserved residues and/or structural features in SECIS function. These might act as signals for interaction with SBP, the SECIS binding protein that we have characterized.

The ternary complex of aminoacylated tRNA and EF-Tu-GTP. Recognition of a bond and a fold

The refined crystal structure of the ternary complex of yeast Phe-tRNA Phe, Thermus aquaticus elongation factor EF-Tu and the non-hydrolyzable GTP analog, GDPNP, revelas many details of the EF-Tu recognition of aminoacylated tRNA (aa-tRNA). EF-Tu-GTP recognizes the aminoacyl bond and one side of the backbone fold of the acceptor helix and has a high affinity for all ordinary elongator aa-tRNAs by binding to this aa-tRNA motif. Yet, the binding of deacylated tRNA, initiator tRNA, and selenocysteine-specific tRNA (tRNA Sec) is effectively discriminated against. Subtle rearrangements of the binding pocket may occur to optimize the fit to any side chain of the aminoacyl group and interactions with EF-Tu stabilize the 3′-aminoacyl isomer of aa-tRNA. A general complementarity is observed in the location of the binding sites in tRNA for synthetases and for EF-Tu. The complex formation is highly specific for the GTP-bound conformation of EF-Tu, which can explain the effects of various mutants.

Native bovine selenocysteine tRNA sec secondary structure as probed by two plant single-strand-specific nucleases

Two single-strand-specific nucleases, discovered in plants, have been used to investigate the secondary and tertiary structures of the native bovine liver selenocysteine tRNA Sec. To check the possible influence of nucleotide modifications on these structures, we compared the results obtained with the fully modified tRNA to the unmodified transcript prepared by in vitro T7 transcription of the Xenopus laevis tRNA Sec gene. We found that the structures in solution of the native tRNA Sec and the transcript are very similar despite some differences in accessibility to the enzymatic probes. Indeed, the modified anticodon-loop of native bovine tRNA Sec, containing 5-methylcarboxymethyluridine (mcm 5U 34) and N6-isopentenyladenosine (i6A37), is less accessible to Rn nuclease than that of the transcript: the intensity of bands representing cuts at A 36 and A 38 is much lower as compared to those of the transcript, whereas no cuts were found at the level of i 6A 37 in the anticodon loop of the native molecule. Surprisingly, the variable arm of the native molecule has been found to be more susceptible to single-strand-specific nuclease action, suggesting a looser structure of the variable arm in native bovine tRNA sec than in the transcript.

Upstream promoter elements are sufficient for selenocysteine tRNA [Ser]Sec gene transcription and to determine the transcription start point

The TATA box, located upstream at about nt -30, and the proximal sequence element, located at about nt -60, are both essential and sufficient for basal level transcription of the Xenopus laevis (Xl) selenocysteine (Sec) tRNA [Ser]Secgene as demonstrated by its microinjection into Xl oocytes. Point mutations within either of these regions abolish transcription, while deletion of the internal boxA element or insertion of 13 nt within the internal boxB element does not impair transcription. The latter mutations (within the internal regions) affect processing of the 3′-trailer sequence. Replacement of the tRNA [Ser]Seccoding sequence with an Escherichia coli M1 RNA gene resulted in expression of the E. coli gene governed by the upstream tRNA [Ser]Secpromoter elements. These studies demonstrate unequivocally that the upstream promoter elements are sufficient for the basal level of tRNA [Ser]Sec gene transcription. Primer extension studies with spacer mutants show that the internal elements do not play a role in selecting the transcription start point ( tsp), but that selection of the tsp is determined by the region upstream from the gene. Further, studies with spacer mutants show that the distance between the TATA box and the tsp is quite likely the critical factor in selecting the position of tsp.

Selenocysteine tRNA and Serine tRNA Are Aminoacylated by the Same Synthetase, but May Manifest Different Identities with Respect to the Long Extra Arm

Selenocysteine (Sec) tRNA [Ser]Sec donates Sec to protein, but interestingly, this amino acid is synthesized on tRNA which is first aminoacylated with serine. Thus, the identity elements in tRNA [Ser]Sec for aminoacylation correspond to elements for seryl-tRNA synthetase recognition. As tRNA [Ser]Sec has low homology to the tRNA Ser isoacceptors, it would seem then that the identity elements in tRNA [Ser]Sec involve (1) very specific sequences, (2) conformational features, and/or (3) different points or domains for tRNA [Ser]Sec:synthetase and tRNA Ser:synthetase recognition. Initially, we confirmed that the same synthetase aminoacylates both tRNAs by showing that a mutant tRNA [Ser]Sec which has a blocked 3′-terminus is a competitive inhibitor of tRNA Ser aminoacylation with a partially purified and a highly purified seryl-tRNA synthetase preparation. The discriminator base (base G73) is essential for aminoacylation of tRNA [Ser]Sec and tRNA Ser, while the long extra arm plays an important role which seems to be orientation- and length-specific in tRNA Ser and, in addition, may manifest sequence specificity in tRNA [Ser]Sec. This difference in the tRNA recognition specificity is discussed. The acceptor stem, DHU stem, and TψC stem contribute to the recogntion process, but to a lesser extent than the discriminator base and the long extra arm.

Eukaryotic selenocysteine inserting tRNA species support selenoprotein synthesis in Escherichia coli.

Although the tRNA species directing selenocysteine insertion in prokaryotes differ greatly in their primary structure from that of their eukaryotic homologues they share very similar three-dimensional structures. To analyse whether this conservation of the overall shape of the molecules reflects a conservation of their functional interactions it was tested whether the selenocysteine inserting tRNA species from Homo sapiens supports selenoprotein synthesis in E. coli. It was found that the expression of the human tRNA(Sec) gene in E.coli can complement a lesion in the tRNA(Sec) gene of this organism. Transcripts of the Homo sapiens and Xenopus laevis tRNA(Sec) genes synthesised in vitro were amino-acylated by the E.coli seryl-tRNA ligase although at a very low rate and the resulting seryl-tRNA(Sec) was bound to and converted into selenocysteyl-tRNA(Sec) by the selenocysteine synthase of this organism. Selenocysteyl-tRNA(Sec) from both eukaryotes was able to form a complex with translation factor SELB from E.coli. Although the mechanism of selenocysteine incorporation into seleno-proteins appears to be rather different in E.coli and in vertebrates, we observe here a surprising conservation of functions over an enormous evolutionary distance.

Promoter Strength and Structure Dictate Module Composition in RNA Polymerase III Transcriptional Activator Elements

RNA polymerase III transcription of genes with external promoters only (e.g. U6 snRNA) or containing in addition an internal B box (selenocysteine tRNA Sec) is stimulated by upstream elements; a distal sequence element (DSE) for U6 or an activator element in the tRNA Sec gene. In contrast to the composite structure of the DSE which requires an octamer motif, the Xenopus tRNA Sec activator element contains an SPH motif only. In vivo transcription is optimally stimulated by SPH in an absolute octamer-independent manner since adding octamer does not induce superstimulation. Experiments performed in the work presented here led to the following observations. Co-operation between SPH and octamer motifs can be detected in two distinct cases: first when these motifs are placed in front of B box-less tRNA Sec or U6 external promoters and second, if either element of the external promoter (proximal sequence element or TATA element), or the SPH motif itself, are altered. Altogether, our data provide evidence that an SPH motif can function alone in an optimized promoter only. In contrast, an octamer becomes indispensable when the basal promoter is weak or disabled. It follows that module composition of Pol III transcriptional activator elements is dependent on the structure and strength of the promoter. This reveals the existence of cross-talk between activator and promoter elements, mediated by the bound transcription factors, which are thus able to compensate for each other in order to allow successful assembly of the transcription complex.