©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Developmental Stage-specific Regulation of Xenopus tRNA Genes by an Upstream Promoter Element (*)

Wanda F.Reynolds

From the(1) San Diego Regional Cancer Center, San Diego, California 92121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Typically the internal promoter elements of tRNA genes are necessary and sufficient to support transcription. Here a sequence element preceding a Xenopus tRNA gene is shown to be required for transcription in late stage, but not early stage oocyte extracts. The constitutive tyrD gene is expressed in both early and late oocyte extracts, whereas the early oocyte-specific tyrCooc gene is only expressed in early extracts. An upstream promoter element (URR), between positions -42 and -14 of the tyrD gene, mediates this differential expression. The URR is required for tyrD transcription in late oocyte extracts. Placing the URR upstream of the tyrCooc gene allows this gene to be transcribed in late extracts. The URR is irrelevant to transcription in early extracts; transcription of tyrD or tyrCooc requires only the internal promoter sequences. This indicates the polymerase III transcriptional machinery changes during oogenesis, resulting in a stringent upstream sequence requirement. Mutations within the URR are shown to alter the preferred site of initiation by RNA polymerase III. Shifting the position of the URR upstream by one-half helical turn also repositioned the site of initiation, suggesting the URR directs the placement of the initiation factor complex or polymerase itself.


INTRODUCTION

The core promoter of tRNA genes consists of two highly conserved internal elements termed the A and B boxes; however, the regulated expression of tRNA genes is in many cases due to upstream sequences (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) . Upstream elements have been found to increase transcriptional activity by facilitating binding of transcription factors or interaction by RNA polymerase III (2, 3, 4) . In some cases, a tissue-specific (6) or species-specific (7) requirement for upstream promoter elements suggests that the polymerase III factor complement can be different in different cell types. This study concerns the promoter sequences which regulate two Xenopus tyrosine tRNA genes: tyrD, which is constitutively expressed in adult somatic cells, and tyrCooc, which is expressed in early oocytes and gradually inactivated as the oocytes mature (9, 10) .

Transcription of tRNA genes requires at least two multisubunit factors, TFIIIC and TFIIIB (for reviews see Refs. 1, 11, and 12). Studies in yeast have provided the most detailed information as to the structure-function relationships of the various subunits of TFIIIC and TFIIIB. The principle binding sites for TFIIIC are the internal A and B boxes. Yeast TFIIIB is comprised of two subunits in association with TATA-binding protein (13) . This TFIIIB complex appears to lack independent DNA binding capability and is recruited through contact with TFIIIC to bind to a 40-bp() region preceding tRNA genes (14) . Within this complex, TFIIIB forms a tight association with the DNA, which is largely sequence-independent. Following high salt removal of TFIIIC, TFIIIB is able on its own to direct initiation by RNA polymerase III, designating TFIIIB as the initiation factor, whereas TFIIIC serves to position that complex upstream of the gene (14) .

Higher eukaryotes have requirements for tissue-specific expression of particular tRNA genes. There are in Xenopus large families of oocyte-specific tRNA (10, 15) and 5 S RNA genes (16) , presumably to allow for large scale production of the RNA products for use in early development. As oocytes mature, these oocyte-type gene families are selectively and stably inactivated, whereas constitutive or somatic-type tRNA and 5 S RNA genes remain active (10, 16, 17, 18, 19) . Partial purification and reconstitution of the polymerase III factors from early and late stage oocytes has indicated that a change in TFIIIC properties is responsible for the selective loss of oocyte type tRNA gene expression (19) . Late stage TFIIIC loses the ability to activate or form stable complexes with oocyte type tRNA promoters. This change in TFIIIC activity can be brought about by the cdc25 phosphatase which is instrumental in initiating the final stages of oocyte maturation (20) .

This study delineates the promoter sequences which are responsible for the regulation of the oocyte type tyrCooc gene and the lack of regulation for the constitutive tyrD gene. The findings show that transcription in late stage oocyte extracts requires a positive sequence element preceding the tyrD gene (between -42 and -14), including the probable binding site for the TFIIIB complex. This upstream regulatory element is developmental stage specific; it has no effect on transcription in early oocyte extracts.


MATERIALS AND METHODS

Xenopus DNAs

Plasmid tyrC is previously described pTyrC* (9) which contains the oocyte-type tRNAtyrC gene and 5`-flanking sequences joined to the 3`-flanking sequences of the tyrD gene. Plasmid pTyrD contains the tRNAtyrD gene as described previously (9) . The tyrD gene with tyrCooc upstream sequence was described previously as p5`C-3`D, and the tyrC* gene with tyrD upstream sequence as p5`D-3`C* (9) . The -42, -30, and -14 deletion mutants of tyrD were made by polymerase chain reaction using synthetic primers starting at the designated positions, combined with a downstream primer starting 20 bp 3` of the tyrD T-stretch termination signal. Polymerase chain reaction primers were also used to make the -42tyrD constructs with triplet base substitutions centered at -39, -36, and -33. Primers started at position -and extended past the triplet mutation sites. For the -14tyrCooc construct, primers were used to place CCTGCACCCACCGG upstream of the tyrC* gene. This sequence is tyrD-type at -14 to -9 (underlined) and tyrC type at -8 to -1 ( bold). All of the polymerase chain reaction-derived constructs are ligated in the same orientation into Bluescript KS+ (Stratagene) at the EcoRV site.

The tyrD maxigene construct was made by insertion of a 200-bp vector DNA fragment at the MspI site 18 bp preceding the T-stretch termination signal. The plasmids containing the Xenopus somatic and oocyte 5 S genes have been described previously (18, 21) .

Oocyte Extract Preparation

For preparation of early oocyte extracts, ovaries were removed from intermediate sized frogs (3-4 inches) and contained a mixture of oocytes at all stages of maturation with relatively few fully mature stage (Dumont stage VI). The ovaries were homogenized and S100 extracts prepared as described previously (20) . For preparation of late stage oocyte extracts, ovaries were removed from large, adult frogs and treated with collagenase. The mature (stage VI) oocytes were isolated and S150 extracts prepared as described previously (20, 22) .

The extracts were fractionated by phosphocellulose chromatography to separate TFIIIB and TFIIIC activities according to previously described procedures (20, 23) , and these fractions combined to form a reconstituted transcription system (19) .

Transcription Reactions

Transcription assays were performed for 3 h (unless otherwise stated in the text) at 22 °C in a reaction volume of 25 µl containing 20 µl of oocyte extract, 5 mM MgCl, 1 mM dithiothreitol, 0.6 mM each ATP, CTP, and UTP, 20 µm GTP, 10 µCi of [-P] GTP, and 100 ng of template plasmid DNA. Reactions were terminated by addition of 5 volumes of 20 mM EDTA, 0.5% sodium dodecyl sulfate (SDS), and carrier RNA. The transcription products were purified by extraction with phenol/chloroform (1:1), followed by ethanol precipitation. The precipitate was redissolved in 95% formamide, 0.05% bromphenol blue, 1 TBE (90 mM Tris base, 90 mM boric acid, 2.5 mM EDTA). The samples were heated at 95 °C for 3 min, and an aliquot was loaded onto 8% polyacrylamide gels containing 1 TBE and 8 M urea. Following electrophoresis, the gel was dried and exposed to x-ray film. Typical exposure times were between 1 and 4 h. Quantitation of the relative amounts of label incorporated was determined by excising and counting the corresponding regions of the gel.


RESULTS

A Sequence Preceding the tyrD Gene Is Essential for Transcription in Late Oocyte Extracts, but Has No Effect in Early Oocyte Extracts

Earlier studies utilized distinct intron sequences to demonstrate that tyrCooc and tyrD are differentially regulated in vivo in early versus late stage oocytes and in somatic cells (9, 10) . A similar pattern of regulation is observed in vitro using oocyte extracts; tyrCooc is transcribed in early, but not late, oocyte extracts, whereas the constitutive tyrD gene is transcribed at high levels in both (20) . An investigation was initiated to identify the DNA sequences responsible for the differential expression in late stage oocytes. The two genes differ in their upstream sequences, at one position within the B box promoter element, and by distinct introns of 10-12 bp situated between the A and B boxes (9) . Previous studies indicated that sequence differences upstream, but within 12 bp of the genes, resulted in 6-fold higher transcriptional efficiency for tyrD, relative to tyrCooc, in somatic cell extracts (9) . To determine which sequences are responsible for the more pronounced 50-100-fold difference in transcription levels in late oocyte extracts, a series of deletion and exchange mutants were compared (Fig. 1 A). These included the tyrCooc gene with 65 bp of native 5` sequence, the tyrD gene with 95 bp of native 5` sequence (constructs 1 and 5), those genes with exchanged upstream sequences (constructs 3 and 7), both genes having 14 bp of tyrD upstream sequence (CCTGCATGAACATC) (constructs 4 and 6), or 8 bp of the tyrCooc immediate upstream sequence (cctgcaCCCACCGG)(constructs 2 and 8). This tyrCooc upstream region (underlined) is highly GC-rich and surrounds the site of transcription initiation at position -5. The position designated +1 represents the first nucleotide of the mature tRNA sequence, rather than the actual site of transcription initiation, in accordance with previous studies. As reported previously, transcription initiation for tyrD occurs at positions -5 and -3, whereas in the case of the tyrCooc gene, initiation occurs at position -5 (10) . All eight constructs have the same termination signal and 3` sequences derived from the tyrD gene.


Figure 1: Sequences preceding the tyrD and tyrCooc genes cause differential expression in late but not early oocyte extracts. A, the following constructs were assayed by in vitro transcription in oocyte extracts: the tyrCooc gene with 65 bp of native upstream sequence (1), 14 bp of tyrD-tyrCooc upstream sequence (2), 95 bp of tyrD upstream sequence (3), or 14 bp of tyrD upstream sequence (4). The tyrD constructs contained 95 bp (5) or 14 bp (6) of native upstream sequence, 65 bp of tyrCooc upstream sequence (7), or 14 bp of tyrD-tyrCooc upstream sequence (8). For constructs 2 and 8, the upstream 14 bp are the tyrD sequence from -14 to -9 (CCTGCA) and the tyrCooc GC rich sequence from -8 to -1 (CCCACCGG). For constructs 4 and 6, the 14 bp upstream are tyrD type (CCTGCATGAACATC). The relative transcriptional activities shown were quantified from the data shown in B. B, in vitro transcription assays were carried out in S150 extracts of early ( upper panel) and late stage oocytes ( lower panel) as described under ``Materials and Methods.'' The reactions were performed for 3 h at 25 °C, and the radiolabeled RNAs were purified, resolved in 8% sequencing gels, and exposed to film. An autoradiograph is shown. The numbers and construct names refer to constructs 1-8 shown in A. C, diagrammatic representation of the data showing the relative contributions of the upstream versus coding sequences to the tyrD transcriptional advantage in late stage extracts. D, graphic display of the data shown in B.



The eight constructs were tested in transcription reactions in extracts of early versus late stage oocytes. In early extracts, the upstream sequences had no significant effect on transcription; all eight constructs were transcribed with similar high efficiency (Fig. 1 B, upper panel, and D). Deletion to position -14 did not alter the level of transcription of either gene, indicating the internal promoter is the primary determinant of transcriptional activity in early oocytes. Surprisingly, this is not the case in late oocyte extracts; only two of the eight constructs produced strong transcriptional signals, those being either gene with 95 bp of tyrD upstream sequences (Fig. 1 B, lower panel, lanes 3 and 5, and D). Deletion of the tyrD sequences between -95 and -14 reduced transcription to nearly undetectable levels ( lanes 4 and 6), indicating this region contains an essential promoter element. The 14-bp sequence immediately preceding the tyrD gene provided a relatively small, 2-3-fold advantage ( lanes 1, 4, 6, and 8). Substitution with the 8 bp of GC-rich tyrCooc sequence surrounding the initiation site abolished that 2-fold advantage ( lanes 2 and 4). These results indicated that tyrD sequences further upstream than -14 are essential for high levels of transcription in late oocyte extracts, with a smaller contribution from sequences within 14 bp of the tyrD gene. These results are in contrast to previous findings using somatic cell extracts which showed the tyrD gene to be only 6-fold more active than tyrCooc and that advantage was shown to be due entirely to the 12 bp of sequence immediately preceding the gene (9, 10) .

The tyrD upstream regulatory region (URR) is a positive effector of transcription. In contrast, the tyrCooc upstream region has no effect on transcription, either positive or negative. Replacing the tyrD sequences preceding position -14 with vector sequences virtually abolished transcription (Fig. 1, B and D, lanes 5 and 6), while replacing the corresponding tyrCooc upstream region with vector sequences did not rescue transcription ( lanes 1 and 2). This is a stage-specific transcriptional enhancement; the upstream sequences have no effect on transcription in early oocyte extracts, yet are essential for transcription in late oocyte extracts, suggesting a change during oogenesis in the transcriptional machinery which interacts with the upstream region. The Positive Regulatory Element Is Situated between -42 and -14 of the tyrD Gene-To determine more precisely which sequences are important for transcription, deletions and mutations were introduced (Fig. 2 A). Deletion to position -42 did not reduce transcriptional activity (Fig. 2, B and D, lanes 1 and 2), indicating the positive element is located between positions -42 and -14. Deletion to position -30 reduced tyrD transcription by 10-20-fold ( lanes 2 and 6), whereas deletion to position -14 reduced the tyrD signal still further, to nondetectable levels ( lane 7), an additional decrease of 5-10-fold. This indicates that sequences extending between positions -42 and -14 are essential for high levels of expression in late stage extracts. The effect is specific to late stage oocytes; deletion to -30 or -14 reduced transcription by no more than 2-3-fold in early oocyte extracts (Fig. 2 D).


Figure 2: The sequence between -42 and -14 of the tyrD gene is required for transcription in late but not early oocyte extracts. A, the sequences preceding the tyrD and tyrCooc genes are shown. tyrD mutant constructs designated -39, -36, and -33 have 42 bp of tyrD upstream sequence with triplet base substitutions indicated by underlined, nonbold type. The -39 and -33 mutants have the corresponding tyrCooc sequence inserted at those positions. The -36 mutant abolishes a conserved AAG sequence present upstream of both tyrCooc and tyrD. The -30 deletion construct has sequences preceding -30 replaced by vector sequences. The -14 deletion construct has sequences preceding -14 replaced by vector sequences. All constructs are inserted in the same orientation and so have the same vector sequences preceding the tyrD sequences. For all constructs, nucleotides in bold are identical to the tyrD upstream sequence. Below is shown the 32 bp of sequences preceding the somatic-type 5 S and oocyte-type 5 S genes. The lines above the sequence indicate features common to tyrD and somatic 5 S, including a purine-pyrimidine asymmetry followed by an AAGTYA motif. B, transcription reactions were carried out in early and late stage oocyte extracts. The constructs used in the reactions are the tyrD gene with 95 bp of upstream sequence, and the constructs shown in A, as listed below each lane. C, a diagrammatic representation of results shown in B indicating the relative importance of the tyrD upstream sequences to transcription in late stage extracts. D, a graphic representation of the data shown in B. The signals obtained with the -95tyrD gene ( lanes 1) for each extract was assigned a relative transcriptional activity of 100%. The relative transcriptional activity shown for lanes 2-8 is relative to that of -95tyrD in the same extract.



The DNA sequence between positions -42 and -14 has features which might affect the ability to form an active transcription complex (Fig. 2 A). The region between -41 and -28 is relatively AT-rich, a characteristic which has been suggested to facilitate bending of DNA (24) , and it is known that the TFIIIB initiation complex produces a sharp bend in DNA (25) . A second feature is purine-pyrimidine asymmetry between positions -42 and -32, in which the upper strand is comprised of 10 out of 11 purine residues. Interestingly, a 14-bp asymmetric sequence is similarly situated between positions -28 and -15 preceding the somatic 5 S gene, whereas upstream of the oocyte-specific 5 S gene, the asymmetry is centrally interrupted at two positions (Fig. 2 A, bottom). Like tyrD and tyrCooc, the somatic and oocyte 5 S genes are transcribed in early extracts, whereas only the somatic 5 S gene is active in late extracts (18, 26) (Fig. 2 B, lanes 9 and 10). Previous studies have shown that 32 bp immediately preceding the somatic 5 S gene is responsible for about half of the 100-fold somatic 5 S transcriptional advantage in late extracts (21) . Therefore there is reason to suspect that similarities in the upstream sequences of tyrD and somatic 5 S may be significant in terms of the selective transcription of these genes in late oocyte extracts.

To investigate the significance of the AT-rich-asymmetric sequence preceding tyrD, a series of triplet base substitutions were introduced between -40 and -32 (Fig. 2 A); AAC centered at -39 was substituted by TTG, the corresponding sequence upstream of tyrCooc; AAG centered at -36, common to both genes, was substituted by pyrimidines, TCC; AAG centered at -33 was substituted by TCC, the corresponding tyrCooc sequence. Each of these triplet substitutions resulted in a 2-3-fold decrease in transcription level (Fig. 2 D, lanes 3-5), indicating this entire region extending from -40 to -32 contributes to the transcriptional enhancement.

The AT-rich/asymmetric regions ends in a motif AAGTYA in the case of both the tyrD and somatic 5 S genes (Fig. 2 A, underlined). An earlier study reported the related motif AAAGT as being present in the 5`-flanking sequences of a number of Xenopus tRNA and 5 S genes and implicated for interaction with a novel stimulatory factor (27) . This motif is present upstream of both the somatic and oocyte 5 S genes (but not tyrD or tyrCooc) and overlaps with the AAGTYA motif (AAAGTYA). The triplet substitution centered at -33 disrupts the AAGTYA motif and results in a 2-3-fold drop in transcription, suggesting this motif may be important for transcription in late oocytes.

As another point of interest, the site of initiation by RNA polymerase III is altered by the -36 and -33 triplet substitutions. This is seen as a change in the relative proportions of two transcript sizes. The -42tyrD gene produced equal proportions of two transcripts corresponding to the two previously reported initiation sites at -3 and -5 (Fig. 2 B, lane 2) (9, 10) . The -36 triplet mutation results in only the larger transcript ( lane 4), whereas the -33 triplet mutant produces the smaller transcript ( lane 5). Since these sequence changes are located at least 25 bp distant from the actual initiation sites, these changes are unlikely to directly affect the choice of initiation residue by RNA polymerase III and instead may be affecting the positioning of the TFIIIB initiation complex. It is important to note that these sequence changes alter the initiation site both in early and late extracts (Fig. 2 B), thus this positioning effect is not specific to late stage extracts.

Prolonged Incubation in the Presence of Transcription Factors Does Not Rescue Templates Lacking the Upstream Regulatory Element

The polymerase III transcription factors form a stable complex with DNA which remains associated throughout repeated rounds of transcription (28, 29) . Certain templates, such as oocyte 5 S, are less able to form stable complexes with transcription factors. However, there is evidence that increased time of incubation with factors allows eventual formation of stable active complexes on the oocyte 5 S gene (30) . To determine if this is the case for templates lacking the URR, the DNAs were incubated in late extracts for increasing periods of time, prior to addition of radiolabeled nucleotide (Fig. 3). The templates were -65tyrC, and tyrD with 42, 30, or 14 bp of native upstream sequence. The times of preincubation were 1 or 2 h, followed by 1 additional h with labeled nucleotide. Even the 2-h preincubation period (3 h total incubation) was insufficient to raise the transcription signals of templates lacking the URR ( lanes 9-12). This indicates that prolonged incubation does not result in the eventual formation of active complexes on templates lacking the URR.


Figure 3: Templates lacking the tyrD upstream promoter remain inactive even after prolonged incubation periods in late stage extracts. In the first set of transcription reactions ( lanes 1-4), the tyrD gene with 42, 30, or 14 bp of native upstream sequences, and the tyrCooc gene were incubated for 2 h in late stage oocyte extracts in the presence of radiolabeled nucleotide. In the second set ( lanes 5-8), the templates were incubated for 1 h in the extract without radiolabeled nucleotide and then the labeled nucleotide was added for 1 additional h. In the third set ( lanes 9-12), the preincubation period was 2 h, and the radiolabeled nucleotide was added for the 3rd h. The RNA products were purified, resolved in gels, and exposed to film as described in Fig. 1.



The tyrD URR Facilitates Stable Binding by Polymerase III Transcription Factors

Template commitment assays provide a means to test the stability of factor complexes. In these assays, one template is preincubated in the extract for 1 h to allow factor binding. A reporter template is then added and incubation continued for 2 h in the presence of labeled nucleotide. The degree to which the reporter template is inhibited provides a measure of the ability of the first DNA to stably sequester transcription factors. This assay relies on the established concept that polymerase III transcription factors, once bound to DNA, remain stably associated for many consecutive rounds of transcription (31) . In the experiment shown in Fig. 4, tyrD constructs having 42 or 14 bp of native upstream sequences were preincubated in intermediate stage oocyte extracts for 1 h, followed by addition of a reporter tyrD maxigene construct. Preincubation with -42tyrD resulted in nearly complete inhibition of the tyrD maxigene ( lanes 1 and 2), indicating that most of the limiting factors were stably sequestered by -42tyrD. In contrast, preincubation with the same amount of the -14tyrD construct resulted in little to no inhibition of the maxigene reporter ( lane 3). These findings indicate the URR is involved in formation of stable factor-DNA complexes.


Figure 4: Sequences preceding tyrD facilitate stable binding by transcription factors. Template exclusion assays were carried out in intermediate oocyte extracts for a total of 3 h with radiolabeled nucleotide. In lanes 2 and 3, 200 ng of -42tyrD or -14tyrD plasmid DNA were preincubated in the extract for 1 h to allow factor binding, followed by addition of 200 ng of a reporter tyrD maxigene, and the reaction continued for 2 additional h. In lane 1, no DNA was present during the preincubation period and then the tyrD maxigene was added for the remaining 2 h of incubation. The RNA products were purified, resolved in a sequencing gel, and an autoradiograph obtained. The tyrD maxigene produces a major transcript of greater than 200 bp and smaller transcripts which probably represent premature termination events.



TFIIIC from Late Stage Oocytes Is Unable to Activate the tyrD Gene Lacking the URR

The factors known to take part in stable complex formation are TFIIIC and TFIIIB. Previous findings have shown that the properties of TFIIIC are different in early and late stage oocytes (19) . Late stage TFIIIC fails to form a stable, active complex with oocyte-specific tRNA genes, while retaining the ability to activate the somatic type 5 S RNA gene. The principle binding site for TFIIIC is the internal B box and A box elements, not the URR region. However, protein-protein contacts with TFIIIC are thought to be essential to direct TFIIIB to bind to sequences in or near the URR (14) . Thus, modification to TFIIIC in late stage oocytes could conceivably alter the interaction with TFIIIB, thereby altering the binding specificity of TFIIIB. To determine if the change in TFIIIC activity is mediated through the upstream sequence, these factors were separated by phosphocellulose chromatography from early and late stage extracts (23) . The resultant fractions are crude, containing many components in addition to classical TFIIIC and TFIIIB activities. Fraction TFIIIC from either stage was reconstituted with fraction TFIIIB from late extracts, which also contains TATA-binding protein and RNA polymerase III. A template lacking the URR ( -14tyrD) was transcribed in the presence of early but not late stage TFIIIC (Fig. 5). The template containing the URR ( -42tyrD) was transcribed with either early or late TFIIIC. In the reverse experiment, with early TFIIIC in all four reactions, the -14tyrD template was transcribed in the presence of either early or late TFIIIB (Fig. 5). These results support a model whereby a change in the properties of TFIIIC, or a component which copurifies in fraction TFIIIC, can affect transcription by a mechanism mediated by the upstream URR.


Figure 5: The tyrD gene lacking the URR is not transcribed in the presence of TFIIIC from late stage oocytes. In vitro transcription reactions in the left panel were carried out in a reconstituted system with partially purified fraction TFIIIB from late oocytes, and TFIIIC from either late ( lanes 1 and 2) or early ( lanes 3 and 4) oocytes. In the right panel, fraction TFIIIC from early oocytes was present along with either late ( lanes 1 and 2) or early ( lanes 3 and 4) TFIIIB. The templates are the -42TyrD construct containing the URR, and the -14tyrD construct lacking the URR.



Repositioning the URR Upstream by One-half Helical Turn Alters the Site of Transcription Initiation

Yeast studies indicate the TFIIIB initiation complex contacts DNA sequences upstream and within 45 bp of tRNA genes (14, 32, 33) . Difficulties in purifying TFIIIB from higher eukaryotes has prevented direct physical determination of the site of TFIIIB binding; however, there is evidence from the Bombyx system that the 40-bp region preceding tRNA genes is involved in TFIIIB binding (24) . To investigate whether the tyrD URR may be involved in the positioning of the initiation complex or RNA polymerase III, a 6-base pair sequence was inserted at position -15, shifting the URR further upstream and to the opposite side of the DNA helix (Fig. 6). Moving the URR to the opposite side of the helix could conceivably interfere with protein-protein contacts between TFIIIB and TFIIIC, which binds to the internal promoter. Shifting the URR upstream by half a turn was found to shift the site of initiation. The two original sites of initiation converged into a single site which was approximately 2 base pairs upstream. Transcription also decreased by severalfold. It is unlikely that the 6-bp insertion at position -15 directly affects the choice of initiation residue by RNA polymerase III, since the insertion is 8-12 bp away from the usual site of initiation. Instead, this suggests that shifting the URR further upstream alters the positioning of the initiation complex, thereby affecting positioning of RNA polymerase.


Figure 6: Insertion of a 6-bp sequence at position -15 shifts the URR one-half turn upstream and alters the site of initiation. The diagram shows the site of insertion of the sequence GAATTC at position -15 preceding the tyrD gene. The arrows show the sites of initiation previously reported for tyrD (A residues at -3 and -5) (10) and below is indicated the approximate site of initiation in the insertion mutant. The -15 insertion tyrD mutant and the -42tyrD construct were transcribed in late stage extracts and the transcription products resolved in sequencing gels as shown below. The -15 insertion construct produced a slightly larger transcript size.




DISCUSSION

These results demonstrate that the differential expression of the oocyte-specific tyrCooc and constitutive tyrD genes is due to a defined upstream sequence element. This upstream regulatory region or URR is situated between positions -42 and -14 of the tyrD gene and is required for assembly of a functional transcription complex in late stage oocyte extracts; deletion of the URR abolished transcription of the tyrD gene, whereas placing this sequence element upstream of tyrCooc restored transcription in late extracts. The tyrD upstream element had no effect on transcription of either gene in early oocyte extracts, indicating the regulatory effect is stage-specific, apparently reflecting a change in the polymerase III transcription machinery during oogenesis.

Template exclusion assays indicate the tyrD URR is important for stable complex formation. The only factors as yet known to participate in stable complex formation are TFIIIB and TFIIIC (23, 28, 29, 31) . By analogy to the yeast system, the URR may include the binding site for the TFIIIB initiation complex (14, 33) . As evidence that the tyrD URR affects positioning of the initiation complex or polymerase III, the insertion of a 6-base pair sequence at -15 shifted the URR one-half helical turn upstream and thereby shifted the site of transcription initiation. Moreover, triplet base substitutions introduced within the URR between positions -32 and -37 altered the preferred site of initiation. These mutations are 27 or more bp upstream of the start site, possibly too distant to directly affect polymerase III site selection, suggesting these base changes alter the positioning of the TFIIIB initiation complex.

These findings are unexpected since for most tRNA type genes, the internal promoter, including the A and B boxes, is necessary and sufficient to direct factor binding and initiation by RNA polymerase III, regardless of the upstream sequences (1, 11) . There are exceptions in insect systems in which upstream sequences are required for transcription along with the internal promoter (6, 8, 24) ; silkgland-specific expression of the Bombyx tRNASG requires an upstream sequence, while a different upstream sequence is required for transcription of the constitutive tRNA gene (6, 24) . The sequence preceding the silkgland-specific tRNASG gene results in a less effective interaction by TFIIIB and polymerase III (34) , indicating that in higher eukaryotes, as in yeast, the 40-bp preceding tRNA genes includes the site of interaction by TFIIIB. In mammalian systems, the region preceding tRNA genes has also been shown to influence factor binding and transcriptional activity (4, 35) . Placement of the Lac repressor binding site at various positions upstream of a human tRNA gene interfered with formation of transcription complexes, presumably through steric hindrance, indicating the transcription complex extends at least 35 bp upstream (35) . The findings reported here are distinctive from earlier studies in that the tyrD URR has dramatically different effects on transcription at two stages of oocyte maturation. In late oocyte extracts, the URR is essential for transcription, whereas in early oocyte extracts, the URR is irrelevant. This indicates that the polymerase III machinery changes during oogenesis with resultant changes in DNA sequence requirements and that the upstream 40 bp plays a central role in the regulation of these tRNA genes.

Studies in yeast have shown that TFIIIB, the initiation factor for RNA polymerase III, binds to the region preceding tRNA genes, and it does so largely in a sequence independent manner (14) . It is thought that TFIIIC binds the internal promoter and directs TFIIIB, through protein-protein contacts, to bind a specified distance upstream of the initiation site. Consistent with those findings, the sequences preceding the XenopustyrC and tyrD genes have no bearing on transcription in early oocyte extracts. Replacement with vector sequences did not affect transcription levels. It is therefore curious that these upstream sequences become essential in late stage oocytes. One possible explanation is that the interaction between factors TFIIIC and TFIIIB is altered in late stage oocytes, such that TFIIIC is unable to direct TFIIIB to bind to DNA in a nonspecific manner, and instead TFIIIB binding requires the URR sequence or its equivalent.

In support of a model invoking an altered interaction between TFIIIC and TFIIIB, a recent study showed that a mutation in a yeast TFIIIC subunit altered its ability to recruit TFIIIB (36) . Our previous studies have shown that the properties of TFIIIC change in maturing oocytes, such that late stage TFIIIC is unable to form an active complex with early oocyte-specific tRNA genes (19, 20) . As shown here, late stage TFIIIC, or a component which copurifies with this activity, requires the tyrD URR in order to support transcription, even though the URR sequence is outside the canonical TFIIIC binding site. This may reflect an ineffective TFIIIC-TFIIIB interaction, due to modification to either TFIIIC (20) or TFIIIB (37) in late stage oocytes. It is also important to point out that the phosphocellulose fraction TFIIIC used in this study is a crude preparation and therefore contains many components in addition to classical TFIIIC activity. It remains a distinct possibility that components other than TFIIIC mediate this regulation and such components might interact directly with the URR.

Certain features of the URR, other than primary nucleotide sequence, may contribute to its positive effect on transcription. This region exhibits high AT content and purine-pyrimidine asymmetry which might affect flexibility or melting properties. The capacity to adopt bends may be important for sequences preceding tRNA genes, since yeast TFIIIB is known to produce a sharp bend in the DNA (25) . High AT content is often associated with the 5`-flanking regions of tRNA genes (7, 24, 32) and is characteristic of certain upstream sequences which have a positive effect on tRNA transcription (3, 24) . In the case of the sequence preceding a Bombyx constitutive tRNAC gene, the AT-rich character is more important than the actual DNA sequence (24) .

The regulation of the tyrCooc and tyrD genes is similar to regulation of the oocyte-type and somatic-type 5 S RNA genes. Both 5 S genes are transcribed efficiently in early oocyte extracts, but the somatic 5 S gene is 50-100-fold more active than the oocyte 5 S gene in late oocyte extracts (18) . This regulation is partly due to upstream sequences within 32 bp of the genes and partly due to internal sequence differences (21, 26) , unlike the tyrCooc and tyrD genes in which the internal sequence differences play no significant role in the differential expression. The mechanisms by which the upstream promoter elements enhance transcription of somatic tyrD and somatic 5 S genes may be related; in the case of yeast 5 S and tRNA genes, TFIIIC binds initially to the internal promoter of both types of genes and thereafter recruits TFIIIB to bind to the upstream regions (14) . Earlier findings have indicated that sequences preceding the Xenopus somatic-type 5 S gene increase binding efficiency for transcription factors (21) , as shown here for tyrD (Fig. 4). These two genes also have common AT-rich and asymmetric sequences in their upstream regions, as shown here (Fig. 2).

In summary, a sequence element preceding the constitutive tyrD gene and encompassing the probable binding site of the TFIIIB initiation complex is required for transcription in late stage oocyte extracts. The absence of this upstream sequence, or a sequence with equivalent properties, is responsible for the transcriptional inactivity of the early oocyte type tyrCooc gene. This apparently reflects changes in the polymerase III transcriptional machinery during oocyte maturation such that a particular upstream sequence becomes essential for formation of an active complex. A suggested model is that modification to TFIIIC alters the interaction with TFIIIB, or other component of the preinitiation complex, such that the complex loses the ability to interact with the upstream DNA in a sequence-independent manner.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant RR09118-09 (National Center for Research Resources program). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: bp, base pair(s); URR, upstream regulatory region.


ACKNOWLEDGEMENTS

We thank Stuart Clarkson for kindly providing plasmid DNAs and Richard Maki for critical comments on the manuscript.


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