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Originally published In Press as doi:10.1074/jbc.M601702200 on March 21, 2006

J. Biol. Chem., Vol. 281, Issue 20, 14321-14329, May 19, 2006
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Mapping the Principal Interaction Site of the Brf1 and Bdp1 Subunits of Saccharomyces cerevisiae TFIIIB*Formula

George A. Kassavetis1, Robert Driscoll2, and E. Peter Geiduschek

From the Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634

Received for publication, February 22, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Brf1 subunit of the central RNA polymerase (pol) III transcription initiation factor TFIIIB is bipartite; its N-terminal TFIIB-related half is principally responsible for recruiting pol III to the promoter and for promoter opening near the transcriptional start site, whereas its pol III-specific C-terminal half contributes most of the affinities that hold the three subunits of TFIIIB together. Here, the principal attachment site of Brf1 for the Bdp1 subunit of TFIIIB has been mapped by a combination of structure-informed, site-directed mutagenesis and photochemical protein-DNA cross-linking. A 66-amino acid segment of Brf1 is shown to serve as a two-sided adhesive surface, with the side chains projecting away from its extended interface with TATA-binding protein anchoring Bdp1 binding. An extensive collection of N-terminal, C-terminal, and internal deletion proteins has been used to demarcate the interacting Bdp1 domain to a 66-amino acid segment that includes the SANT domain of this subunit and is phylogenetically the most conserved region of Bdp1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The RNA polymerase (pol)3 III transcription apparatus of yeast comprises, in addition to the polymerase itself, three transcription initiation factors, TFIIIA, -B, and -C.

The three-subunit TFIIIB is the core initiation factor required and sufficient for recruiting pol III to the promoter and accurately initiating transcription. The large six-subunit TFIIIC places TFIIIB on DNA upstream of the transcriptional start site and protects the generally very small pol III transcription units from encroachment by repressive chromatin (1, 2). TFIIIA is the 5 S rRNA gene-specific factor that places TFIIIC on the transcription unit-internal promoter of these genes (reviewed in Refs. 3–5).

The work that is presented here deals with the structure of TFIIIB, which is composed of three subunits: the TATA-binding protein (TBP), Brf1, and Bdp1. TBP and Brf1 bind tightly and co-purify, whereas Bdp1 dissociates during later steps of conventional column chromatography. (The two separated fractions of TFIIIB have been designated B' and B'', and B double prime has been adopted as the eponym of the Bdp1 subunit.) The 596-amino acid Brf1 is a fusion protein, with an N-terminal half that is related to the pol II transcription initiation factor TFIIB and a pol III-specific C-terminal half. A conserved segment of the C-terminal half (homology domain II; amino acids 439–515) constitutes the Brf1 high affinity binding site for the evolutionarily highly conserved core of TBP (comprising all but the N-terminal 60 amino acids of the Saccharomyces cerevisiae protein); the N-terminal half of Brf1 engages TBP through a separate lower affinity interaction.

The N- and C-terminal halves of Brf1 also provide separate Bdp1-docking sites, with the C-proximal homology domain II contributing the higher Bdp1 affinity. TBP makes only a minor direct contribution to bringing Bdp1 into TFIIIB. Thus, the pol III-specific half of Brf1 appears to contribute the interactions that principally hold TFIIIB together. Indeed, when TFIIIB-promoter complexes are assembled in vitro with the separated halves of Brf1, the DNA complex formed with the Brf1 C-terminal half is stable but transcriptionally almost inactive, whereas the promoter complex formed with the Brf1 N-terminal half is quite unstable but transcriptionally highly active, yielding nearly wild-type levels of transcription (6).

The determination of the structure of a ternary complex formed by a DNA fragment with a strong TATA box, TBP core (amino acids 61–240), and a large C-terminal fragment of Brf1 showed the latter making essentially continuous contact along both lobes of the convex surface of TBP and also interacting with a side and stirrup of the N-terminal TBP lobe (7). The structure also suggested that it might be possible to separate the N- and C-terminal domains of Brf1 and reconnect them through TBP. Indeed, a triple fusion protein that reconfigures TFIIIB topology by placing the TBP core between the N- and C-proximal domains of Brf1 fully replaces both Brf1 and TBP for TFIIIC-dependent and -independent transcription in vitro. Together with Bdp1, this Brf1-TBP fusion protein forms an extremely stable TFIIIB·DNA complex whose footprint is indistinguishable from that of the wild type TFIIIB·DNA complex. The Brf1-TBP triple fusion is also able to replace Brf1 in vivo (8).

Although Bdp1 is an essential yeast protein, removal of large segments from the N- and C-ends is compatible with some level of viability. The strongest conservation of sequence is confined to a single SANT domain located in the C-terminal one-third of Bdp1 (the three-helix SANT domains are deployed as DNA interaction as well as protein interaction modules), and weak conservation extends to either side of this element, over most of the C-terminal half. Not only is the participation of Bdp1 essential for recruitment of pol III to the promoter (presented as double-stranded DNA) by TFIIIB, but it also plays an essential role in initiating promoter opening (9).

In this work, we have exploited the structure of the Brf1(439–596)·TBP core(61–240)·DNA ternary complex for a systematic site-directed mutagenesis that identifies individual residues constituting the primary Bdp1-binding site of Brf1. The result that emerges from the analysis is that Brf1 homology segment II serves as a two-sided adhesive for fixing Bdp1 to TBP. A large collection of C-terminal, N-terminal, and internal deletion variants of Bdp1 has been used to show that its SANT domain (amino acids 415–472) is the Bdp1 segment that interacts with the C-terminal domain of Brf1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Templates and Probes—The linear 366-bp transcription template based on pU6LboxB was prepared as described (10). Three photochemical cross-linking probes placing a photoactive nucleotide in the nontranscribed strand at bp -39/-38, -28, and -13/-12, respectively, were prepared by primer extension with 5-[N'-(p-azidobenzoyl)-3-aminoallyl]dUTP essentially as described previously (11) with the transcribed strand of the 57-bp SNR6 electrophoretic mobility shift assay probe (bp -56 to +1) described in Ref. 8 serving as template. The nontranscribed strand sequence of this DNA from bp -40 to -10 is TUUcGGCTACTAUaTATACGTGTTTTTUUcG where U represents sites for placement of [N'-(p-azidobenzoyl)-3-aminoallyl]dUMP, and lowercase letters indicate the adjacent labeled nucleotides of the respective probe.

Proteins—RNA polymerase III was purified as described (12) (mono Q fraction) and is expressed as fmol of enzyme active for specific transcription. All other proteins are expressed as fmol of protein. The TBP(61–240) core domain (designated TBPc) was generously provided by Z. S. Juo (Stanford University). Bdp1, Bdp1(138–594), Bdp1(263–594), and the Bdp1 internal deletion mutants (all C-terminally His6-tagged) were constructed and purified under native conditions on Ni2+-nitrilotriacetic acid-agarose, BioRex 70, and Superose 12, as described (13); Bdp1(263–594) was purified on Ni2+-nitrilotriacetic acid-agarose only. Bdp1(138–506), Bdp1(138–493), Bdp1(138–477), and Bdp1(138–465) were generated by PCR amplification with Pfu DNA polymerase of the pET21d-Bdp1(138–594) clone with the T7 promoter primer and a mutagenic primer inserting an XhoI site at the appropriate location (primer sequences are available upon request) for reinsertion into pET21d as an NcoI-XhoI fragment. All clones were sequenced, and proteins were purified as cited above for Bdp1(263–594). Bdp1(1–352) and Bdp1(352–594) (designated Bdp1n and Bdp1c, respectively; both N-terminally His6-tagged) were purified under native conditions (14). Brf1 (N- and C-terminally His6-tagged) (6), Brf1(1–365), and Brf1(1–382) (N-terminally His6-tagged (15); the latter is designated Brf1n) were purified under denaturing conditions, as cited for each protein.

Brf1(439–596) (designated Brf1c), TBP(61–240)-Brf1(439–596) fusion (designated TBPc-Brf1c), Brf1(1–382)-TBP(61–240) fusion (designated Brf1n-TBPc), and Brf1(1–382)-TBP(61–240)-Brf1(439–596) (designated Brf1n-TBPc-Brf1c) were constructed (each C-terminally His6-tagged and with an additional C-terminal FLAG tag for Brf1n-TBPc) and purified under native conditions as described (8) (Brf1n-TBPc purification followed that of Brf1n-TBPc-Brf1c to the Ni2+-nitrilotriacetic acid-agarose stage). Proteins were quantified by SDS-PAGE relative to Coomassie Blue-stained bovine serum albumin standards. Clustered multiple and single amino acid substitutions in Brf1 were generated by recombinatory PCR with the pET21d-Brf1c clone cited above, using complementary mutagenic primers separately with the appropriate T7 promoter or T7 terminator vector primer and Taq DNA polymerase. The two DNA amplification products were gel-purified and combined for amplification/recombination with a Taq/Pfu DNA polymerase mixture and the T7 promoter and terminator primers. The resulting product was cloned as a SacII-XhoI fragment into the expression vector originally harboring Brf1(439–497) (see below) and sequenced. The SacII-XhoI fragment from this clone was then transferred to the pET21d-TBPc-Brf1c expression vector originally harboring TBPc-Brf1(439–497). C-terminal truncations of Brf1c were generated as described above for Bdp1(138–506) with the pET21d-Brf1c clone as template followed by the SacII-XhoI fragment replacement of the original clone. The C-terminal truncation clones were sequenced and transferred as a SacII-XhoI fragment to replace Brf1c in the original pET21d-TBPc-Brf1c construct. Mutagenic primer sequences are available upon request. The resulting Brf1c and TBPc-Brf1c mutant proteins were purified as described (8).

Transcription—Protein-DNA complexes were formed at 20 °C for 60 min in 20 µl of Reaction Buffer (40 mM Tris-Cl, pH 8.0, 7 mM MgCl2, 3 mM dithiothreitol, 100 µg/ml bovine serum albumin, 6–8% (v/v) glycerol, 5 µg/ml poly(dG-dC)·poly(dG-dC)), and 50 mM (Fig. 2B) or 70 mM (Fig. 5) NaCl with 60 fmol of the 366-bp U6LboxB DNA, 10 fmol of pol III (added after a 40-min incubation for Fig. 2B), and the quantities of Bdp1, TBPc-Brf1c, and Brf1n or Brf1(1–365) specified in the figure legends. Five µl of a nucleotide mixture (1 mM ATP, GTP, and CTP and 125 µM [{alpha}-32P]UTP (10 cpm/fmol)) in Reaction Buffer was added for 30 min of multiple round transcription. Reactions were stopped, and samples were processed for denaturing gel electrophoresis as previously described (16).

Protein-DNA Photochemical Cross-linking—Protein-DNA complexes were formed at 20 °C for 60 min in 20 µl of the Reaction Buffer specified above (without bovine serum albumin and with 2-mercaptoethanol in place of dithiothreitol) and 50–70 mM NaCl. Reaction mixtures contained 5 fmol each of the -39/-38 and -13/-12 probes (Figs. 2A,3,4A,6(C and D), and Table 1) or 3 fmol each of the -39/-38, -28, and -13/-12 probes (Fig. 6, A and B) and the proteins specified in the figure legends. Following the incubation, 150 ng of poly(dA-dT)·poly(dA-dT) was added, and each sample was UV-irradiated for 7 min. Nuclease treatment prior to SDS-PAGE followed (17).


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  		TABLE 1
Effects of single amino acid substitutions in TBPc-Brf1c on mean Bdp1 cross-linking efficiencies

{dagger} Mean Bdp1 cross-linking efficiencies ±S.D. for each TBPc-Brf1c variant are compared relative to the reference TBPc-Brf1c (corresponding to Ri · Qi and Ro · Qo under "Materials and Methods," respectively). The number of independent determinations is specified in parentheses. Cross-linking efficiency values are in bold if Ri · Qi ≤ 0.5 Ro · Qo, underlined if Ri · Qi + S.D. < 0.5 Ro · Qo, and in cells shaded gray if Ri · Qi < 0.3 Ro · Qo

* Residue numbers refer to positions in full-length Brf1 (i.e. Asn441 is Asn195 in TBPc-Brf1c)

 
Cross-linking efficiencies of unaltered and mutant TBPc-Brf1c proteins were derived from the composite mean of all cross-linking experiments regardless of Bdp1 and TBPc-Brf1c concentrations, since the concentration of TBPc-Brf1c was saturating, and titrations of Bdp1 indicated negligible competition with TBPc-Brf1c for cross-linking. The phosphor image intensity for the DNA adduct of each mutant TBPc-Brf1c variant (Ei) was initially normalized to that of reference type TBPc-Brf1c (Eo) in the individual experiment (ri = Ei/Eo). The following method was employed in order to remove the compounding effect of variation in Eo, the cross-linking signal for reference type TBPc-Brf1c: 1) the mean values of the normalized cross-linking efficiency for the reference type protein (Eo) and each variant (Ei) was determined from all experiments; 2) the ratio of each TBPc-Brf1c variant and reference type value in individual experiments to the cross-experiment mean (Po = Eo/Eo; Pi = Ei/Ei) was determined; and 3) the average, P, of all of the Pi and Po values for an individual experiment, taken together, was then used to renormalize ri for each individual experiment (i.e. Ri = ri/P), and averages of Ri (i.e. Ri) were taken. The same procedure was used to renormalize Bdp1/TBPc-Brf1c cross-linking ratios between experiments for each variant protein (Qi). Finally, the Bdp1 cross-linking efficiency of each variant protein was calculated as the product of its renormalized Bdp1/TBPc-Brfc cross-linking ratio (Qi) multiplied by its mean renormalized TBPc-Brf1c cross-linking efficiency (Ri).

Nomenclature—Protein fusions as well as photochemically generated protein-DNA cross-links are designated by a hyphen (e.g. TBPc-Brf1c); noncovalent association between proteins or between proteins and DNA is designated by a dot (e.g. the TBP·Brf1·DNA complex).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The recently determined structure of the ternary complex formed by Brf1c, TBPc, and DNA, in which Brf1 residues 437–506 were resolved (7), provides a framework for using site-directed mutagenesis to identify Brf1c residues that are important for Bdp1 interaction. We anticipated one possible complication in interpreting the outcome of this systematic amino acid substitution screen; the Brf1c amino acid 439–506 segment interface with TBP is notably extensive and spread out (Fig. 1A), and the stability of at least some secondary structure features of Brf1c (e.g. helix H22) may depend on interaction with TBP (7). Thus, diminished Bdp1 binding by Brf1 mutants may not be solely due to the loss of direct Brf1·Bdp1 contacts but can also arise because of effects on the proper folding and/or alignment of Brf1c along its extensive TBP interface.


Figure 1
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  		FIGURE 1.
Model of Brf1(439–596) linked to TBP(61–240) (TBPc-Brf1c) and of the mutations generated within the resolved segment of Brf1. A, a model of the TBPc-Brf1c fusion. TBPc (cyan ribbon), the resolved Brf1(439–506) segment (light green ribbon) and DNA (sticks) are from Ref. 7. A (GS)6 linker (a possible conformation is shown in orange) wasusedtoconnectTBPresidueMet240 (space-filled, cyan) to Brf1 residue Pro439 (space-filled, light green). The view displays the transcriptional start site-proximal face of this complex. B, Brf1 residues in the resolved segment that were mutagenized are space-filled and color-coded as in C. Views of the convex surface and N-terminal side of TBP are shown. Rotations from the view shown in A are indicated at the left. C, sequence of the resolved segment of Brf1. Amino acids whose side chains contact TBP are in lowercase. Mutagenized residues are colored (Leu and Ile (yellow), Asn and His (green), Thr and Ser (orange), Asp and Glu (red), and Lys and Arg (blue)), with the substituted amino acid shown below the sequence, and the location of Brf1 {alpha} helices H21 to H25 is indicated.

 
To circumvent this possible complication, we have tethered Brf1c to TBPc with the flexible 12-amino acid connector (GS)6, taking advantage of the fact that the C terminus of TBPc (M240; space filled in cyan in Fig. 1A) lies within ~20 Å of the N terminus of Brf1c (P439; space filled in green in Fig. 1A). TBPc-Brf1c, with a hypothetical orientation of the connector (in orange) is shown in Fig. 1A. The high local concentration afforded by this tether and its flexibility should diminish the sensitivity of Brf1c·Bdp1 binding to adventitious effects of mutations on Brf1c·TBPc binding. A similar approach was used to rescue the TFIIA binding defect of a TBP mutant protein in yeast (18). The ability of TBPc-Brf1c to assemble Bdp1 onto DNA and to function for transcription has been documented (8) and is further analyzed below.

A remarkable 29 of the 67 amino acid residues of Brf1c that are resolved in the structure of the ternary Brf1·TBP·DNA complex have side chains that hydrogen-bond to or form van der Waals contacts with residues of TBP (sequence in Fig. 1C, lowercase residues) (7)). Twenty-six of the 38 amino acids whose side chains are not in contact with TBP were substituted, along with Asp458 and Glu484, which make limited van der Waals contacts with TBP, and Asp460 and Asn471, which form hydrogen bonds with TBP in at least two of the four crystallographically independent complexes (mutated residues are highlighted in Fig. 1C). The large aliphatic side chains of Leu and Ile (yellow in Fig. 1B) and large polar side chain of Asn and His (dark green) were replaced with the small aliphatic side chain of Ala; small polar residues Thr and Ser (orange) were replaced with Leu; acidic residues Asp and Glu (red) were replaced with Lys; basic residues Lys and Arg (blue) were replaced with Glu (Asp for Arg502). The effect of mutations on the Bdp1·Brf1 interaction was examined by photochemical protein-DNA cross-linking. The same method has been used previously to identify radical sequence substitutions in TBP that interfere with binding to the TFIIB-related N-terminal half of Brf1 in a DNA complex (19).

We first examined the potential contribution to Bdp1 binding of the amino acid 507–596 segment of Brf1c, which is not resolved in the ternary complex structure. The 51 C-terminal Brf1 residues (546–596) have been shown not to provide a major Bdp1-binding site (6). Two additional C-terminal truncations of Brf1c were generated in the TBPc-Brf1c fusion protein, one ending at amino acid 511 and the other at amino acid 497, within the Brf1 helix H25 (Fig. 1C).

DNA complexes were formed with Bdp1 and TBPc-Brf1c (or its derivatives) and a mixture of two probes covering the SNR6 (U6 snRNA) gene promoter region (bp -56 to bp +1, the start site of transcription), one with the photoactive thymidylate analogue 5-[N'-(p-azidobenzoyl)-3-aminoallyl]dUMP (20) at bp -39 and -38 and with [{alpha}-32P]-dCMP at bp -37 on the nontranscribed strand to monitor the presence of Bdp1, and the other, with [N'-(p-azidobenzoyl)-3-aminoallyl]dUMP at bp -13 and -12 and [{alpha}-32P]dCMP at bp -11 to monitor the presence of TBPc-Brf1c. Fig. 2A shows a photochemical cross-linking experiment with Bdp1 and "full-length" TBPc-Brf1c (i.e. extending to the C terminus of Brf1) or truncated at Brf1 residue 511 or 497. The concentration of TBPc-Brf1c (20 nM) was saturating for DNA cross-linking, but Bdp1 (20 nM) was limiting for assembly onto the TBPc-Brf1c·DNA complex. Truncation of TBPc-Brf1c to amino acid 511 or 497 did not appreciably impair the assembly of Bdp1 (A; quantified below each lane), even at a 2-fold lower Bdp1 concentration (data not shown). Truncation to amino acid 511 or 497 decreased the DNA cross-linking efficiency of TBPc-Brf1c (lanes 3 and 4) but not to the extent observed in the absence of TBPc-Brf1c tethering (data not shown). As noted previously (6, 21), TBPc alone was capable of assembling Bdp1 to a small extent (lane 1).

Brf1(1–509) has been shown to be severely defective in TFIIIC-dependent and -independent transcription and, to a lesser extent, in TFIIIB·TFIIIC·DNA complex formation (22). In contrast, Brf1(1–545) displayed at most a 2-fold defect in TFIIIC-independent transcription of SNR6 as supercoiled DNA (6). Since TFIIIC-independent transcription of supercoiled templates by pol III is substantially independent of the C-terminal half of Brf1 (6), we examined the effect of C-terminal truncations of TBPc-Brf1c on transcription of an SNR6-derived template presented as linear DNA. This transcription requires both the N-terminal (TFIIB-related) half of Brf1 and TBPc-Brf1c (8). The ability of TBPc-Brf1c truncated to Brf1 residue 564, 544, 511, or 497 to support TFIIIC-independent transcription is shown in Fig. 2B (the quantification of two experiments is shown below each lane). Truncation of Brf1c to residue 564 or 544, removing Brf1 homology domain III (amino acids 570–595) (23) diminished transcription 4–5-fold (lanes 1–3; note that TBP binds to the SNR6 TATA box in either orientation, yielding divergent transcripts r-U6 and l-U6). Cross-linking experiments similar to Fig. 2A verified that TBPc-Brf1(439–544) was competent to assemble Bdp1 (data not shown) and not inadvertently inactive. Further truncation of TBPc-Brf1c to residue 511 or 497 reduced transcription to barely detectable levels (lanes 4 and 5). Thus, the 100 C-terminal amino acids of Brf1 (which include homology region III) do not contribute significantly to Bdp1 assembly, but removing them progressively impairs transcription of linear DNA.


Figure 2
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  		FIGURE 2.
Brf1 amino acids 498–596 are dispensable for Bdp1 assembly but are required for transcription of linear DNA. A, protein-DNA photochemical cross-linking assay of Bdp1 binding to TBPc·DNA and TBPc-Brf1c·DNA complexes. Brf1c was C-terminally truncated to amino acid 497 or 511 or left intact (596) as indicated above each lane. All proteins were present at 20 nM concentration. Cross-linked Bdp1 and TBPc-Brf1c are identified at the left. Quantification of Bdp1 and TBPc-Brf1c cross-linking are shown below each lane. Bdp1 and truncated TBPc-Brf1c phosphor image intensities were normalized to the corresponding values for full-length TBPc-Brf1c(439–596). B, transcriptional activity of C-terminal truncation variants of TBPc-Brf1c. Multiple round transcription of a 366-bp DNA fragment derived from pU6LboxB (41) with 200 fmol each of Bdp1, Brf1(1–365), and full-length TBPc-Brf1c or TBPc-Brf1c C-terminally truncated to amino acids 564, 544, 511, or 497, as indicated above each lane. The two divergent transcripts specified by the SNR6 TATA box (r-U6 and l-U6) and a radioactive DNA recovery marker (r.m.) are identified at the left. Quantification of two independent experiments is shown below each lane along with S.D. values (sd). The l-U6 and r-U6 image intensities relative to the recovery marker were combined and normalized to transcription obtained with full-length TBPc-Brf1c. The apparent transcription level obtained with TBPc-Brf1c(439–497) is actually due to background from the recovery marker.

 
We next examined the effect of amino acid substitution between Brf1 residues 441 and 508 on Bdp1 assembly using a set of 10 TBPc-Brf1c variants containing locally clustered mutations. As with the C-terminal TBPc-Brf1c truncations in Fig. 2, the ability of these TBPc-Brf1c cluster mutant proteins to assemble Bdp1 onto DNA was assessed by cross-linking with limiting concentrations of Bdp1 (5 and 10 nM) and saturating concentrations of each mutant TBPc-Brf1c variant (≥20 nM) for both TBPc-Brf1c-DNA and Bdp1-DNA cross-linking. A sample cross-linking analysis is shown in Fig. 3A. Two TBPc-Brf1c cluster mutant proteins (K481E/L482A/R485E (lane 8) and L482A/I486A/L490A (lane 9)) were seen to be clearly defective in assembling Bdp1 when compared with unmutated ("reference") TBPc-Brf1c (lane 2).

The effects of these clustered mutations on Bdp1 assembly were examined more closely by quantifying and normalizing the results of multiple cross-linking experiments with 5 or 10 nM Bdp1 (Fig. 3B). The cross-linking efficiency of Bdp1 was normalized to that of TBPc-Brf1c in the same reaction mixture in order to compensate for differences in phosphor image exposure and probe-specific activity between experiments. These values were then compared with the Bdp1/TBPc-Brf1c cross-linking ratio of the reference TBPc-Brf1c protein (Fig. 3B, lower plot). Although the concentration of each mutant TBPc-Brf1c variant was saturating for cross-linking to DNA, as already specified, the efficiencies of cross-linking consistently differed from that of the reference protein in the same experiment (Fig. 3B, upper plot), with the mutant TBPc-Brf1c proteins generally cross-linking more efficiently. The mean Bdp1 cross-linking efficiencies shown in Fig. 3C compensate for the different cross-linking efficiencies of TBPc-Brf1c variants (as specified under "Materials and Methods").

The quantitative analysis confirmed that the K481E/L482A/R485E and L482A/I486A/L490A mutants were the most defective in binding Bdp1, with relative efficiencies of DNA cross-linking that were at most one-fourth that of the reference protein (Fig. 3C). Of the remaining cluster mutant proteins, N441A/H443A/L444A cross-linked at less than 50% efficiency relative to the reference protein, and so did mutant protein E468K/N471A in the presence of 5 nM Bdp1. The charge reversal variants spanning amino acids 497–508 were also significantly deficient at this lower concentration of Bdp1. The absence of an effect of truncating the Brf1 C terminus to residue 497 on Bdp1 assembly (Fig. 2A) suggests that the ~2-fold decrease in Bdp1 assembly with clustered charge reversal mutations between Brf1 residues 497 and 508 may be due to indirect effects. For example, Lys501 and Lys504 of helix H25 are oriented toward and lie close to (within 5–9 Å of) DNA (7), and Bdp1 assembly may alter the path of this helix to allow contact with downstream DNA sequence (consistent with the creation of an additional DNA bend between the TATA box and the start site of transcription upon Bdp1 entry into the TFIIIB·promoter complex (24)). Alternatively, the highly acidic or basic patches that are generated by these clustered charge reversal mutations (Fig. 1C) may disrupt helix H25.

The clearly defective Bdp1 assembly of the two cluster mutant TBPc-Brf1c proteins altering side chains of residues 481–490 encouraged us to define the Brf1 site for Bdp1 binding more closely by screening a large collection of proteins with single amino acid substitutions. An example of a cross-linking experiment with limiting (10 nM) Bdp1 is shown in Fig. 4A. (The concentration of each TBPc-Brf1c single mutation variant, ≥20 nM, was saturating for DNA cross-linking; data not shown.) Variants L482A and I486A were clearly the most defective in DNA-Bdp1 cross-linking, consistent with the Bdp1-binding region defined by cluster mutants K481E/L482A/R485E and L482A/I486A/L490A. Experiments similar to panel A were performed with 5, 10, and 20 nM Bdp1 (generally with 3–6 replicates at each concentration) and quantified. As also observed with the TBPc-Brf1c cluster variants, the cross-linking efficiencies of the single mutation proteins exceeded that of TBPc-Brf1c (supplemental Fig. S1A). These values were used to derive Bdp1 cross-linking efficiencies from Bdp1/TBPc-Brf1c ratios (supplemental Fig. S1B) that are summarized in Table 1. The error margins of the derived Bdp1 cross-linking efficiencies were such that only differences between a single mutant variant and the reference TBPc-Brf1c exceeding a factor of 2 were taken to be significant. In the presence of 20 nM Bdp1, only TBPc-Brf1c variants L482A and I486A were clearly defective in Bdp1 assembly according to this criterion; at lower concentrations of Bdp1, TBPc-Brf1c variants L482A, K485E, I486A, I488A, and L490A were also impaired. It is noteworthy that Leu482, Ile486, and Leu490 lie on one exposed side of Brf1c helix H24 (Fig. 4B) on the same face as and proximal to TBP Glu93 (~8 Å apart), for which the radical substitution mutation E93R was found to impair Bdp1 assembly (25). TBPc-Brf1c variants N441A, H443A, and L444A were also defective in Bdp1 assembly at lower concentrations of Bdp1 (5 and 10 nM; Table 1) and modestly defective at 20 nM Bdp1. These Brf1 residues lie above the convex surface of the N-terminal lobe of TBP (Fig. 4B).


Figure 3
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  		FIGURE 3.
Defective Bdp1 assembly by TBPc-Brf1c mutant proteins with locally clustered mutations. A, protein-DNA cross-linking assay of Bdp1 binding to TBPc·DNA (lane 1) and TBPc-Brf1c·DNA complexes (lanes 2–12). Clustered Brf1c mutations are identified above lanes 3–12; unaltered TBPc-Brf1c serving as the reference type is shown in lane 2. TBPc and TBPc-Brf1c were present at 20 nM, and Bdp1(138–594) was present at 10 nM. Cross-linked products are identified at the left. The asterisk marks a minor cross-linking product that was not dependent on the presence of Bdp1 and was not included in the quantification of Bdp1 cross-linking. B, quantification of relative TBPc-Brf1c cross-linking to DNA (upper plot) and of the ratio of Bdp1-DNA to TBPc-Brf1c-DNA cross-linking (lower bar graph). The reference type TBPc-Brf1c and the cluster mutant TBPc-Brf1c variants are identified below each bar with the corresponding lane in A. The mean TBPc-Brf1c cross-linking efficiencies of 7–9 replicates (Ri, defined under "Materials and Methods") with S.E. values are shown in the upper plot. The mean Bdp1/TBPc-Brf1c cross-linking ratios from reaction mixtures containing Bdp1(138–594) at 10 nM ({blacksquare}; 3–7 replicates but only 2 for E506K/D508K) or at 5 nM (·; 3–6 replicates) (Qi, defined under "Materials and Methods") with S.E. values are shown in the lower graph. C, Bdp1 cross-linking efficiencies (Qi·Ri, specified under "Materials and Methods") derived from the mean values shown in B. Normalization and other experimental details are described under "Materials and Methods."

 


Figure 4
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  		FIGURE 4.
Single amino acid substitutions in TBPc-Brf1c that decrease Bdp1 assembly. A, a sample cross-linking assay with 10 nM Bdp1(138–594) and 40 nM TBPc-Brf1c reference type (rt) and single mutant variants (identified above each lane). B, model of TBPc-Brf1c identifying Brf1 mutations that diminish Bdp1 assembly (space-filled and color-coded as in Fig. 1). A previously identified residue in TBP that is important for Bdp1 binding is also highlighted (Glu93, in cyan (25)).

 
These results indicate that Bdp1 binds to at least two separate segments of Brf1c, accounting for the fact that none of the TBPc-Brf1c cluster mutant variants reduced Bdp1 assembly to the TBPc-alone base line (Fig. 3A). Among the remaining TBPc-Brf1c single mutation variants, D449K, E463K, E468K, L495A, K504E, and E506K interfered less severely with Bdp1 assembly when Bdp1 was present at 5 and 10 nM. Although the mean Bdp1 cross-linking efficiencies of 9 of the other 12 single mutation TBPc-Brf1c variants (K454E, D460K, N471A, E477K, E478K, K481E, E499K, K501E, and R502D) did not deviate from the reference TBPc-Brf1c sufficiently to be individually significant, they yielded mean Bdp1 cross-linking efficiencies that almost uniformly ranged between 40 and 80% of the reference TBPc-Brf1c efficiency at all Bdp1 concentrations (Table 1). The consistency of these independent observations (involving nine variant proteins, each examined at three Bdp1 concentrations) specifies that additional sites in the extended stretch of Brf1 between the amino acid 441–443 and 482–490 clusters (Fig. 4B) make dispersed and smaller contributions to Bdp1 assembly.

We also examined the effects of the Brf1c cluster mutations on transcription of linear DNA. Since TFIIIB complexes formed with Bdp1, TBP, and only the N-terminal half of Brf1 are competent for transcription of supercoiled SNR6-derived templates (6) and since the transcription of linear DNA requires Brf1 residues C-terminal to amino acid 511 that have little effect on Bdp1 assembly (Fig. 2), it was not clear whether defects in Bdp1 assembly of clustered TBPc-Brf1c mutations would be reflected in transcription. The N441A/H443A/L444A mutations had no effect on transcription (Fig. 5), but an ~2-fold decrease in transcription was generated by K481E/L482A/R485E and L482A/I486A/L490A, and an ~4-fold reduction was generated by K501E/R502D/K504E and E506K/D508K. The last two TBPc-Brf1c variants are at most modestly affected in Bdp1 assembly (Fig. 3C), suggesting that these changes interfere with pol III assembly into the preinitiation complex or at a subsequent step of the transcription cycle.

The SANT Domain of Bdp1 Is Responsible for Brf1c Interaction— Bdp1 can be split at amino acid 352. Bdp1(1–352) (Bdp1n) and Bdp1(352–594) (Bdp1c) individually support TFIIIC-independent transcription of supercoiled DNA in complexes formed with intact Brf1 and TBP (14). Both halves together functionally replace intact Bdp1 in vivo (26). We used split Bdp1 in combination with TBPc-Brf1c and Brf1n-TBPc (a fusion protein linking Brf1(1–382) directly to the N terminus of TBP core) to determine whether the interactions between Bdp1 and Brf1 can be partitioned to their N- and C-terminal halves by means of protein-DNA cross-linking. A detailed rationalization for using the same cross-linking assay in this context is provided in the supplemental material.

In this experiment (Fig. 6A), the triple fusion protein Brf1n-TBPc-Brf1c (8) served as the reference. Brf1n-TBPc-Brf1c assembled Bdp1n and Bdp1c separately (lanes 3 and 4, respectively) and together (lane 2) with comparable efficiency. Only a weak background of Bdp1n cross-linking was detected when Brf1n-TBPc-Brf1c was replaced with TBP core (lane 1); this may signify that TBP-Bdp1 interaction is primarily mediated by the N-terminal half of Bdp1. TBPc-Brf1c, on the other hand, was competent in assembling Bdp1c into the DNA complex (lane 7), whereas the level of cross-linking of Bdp1n (lane 6) was indistinguishable from that of TBPc alone (lane 1). No increase in Bdp1n and Bdp1c cross-linking was observed when both halves were present (lane 5). Adding Brf1n to the reaction mixture containing TBPc-Brf1c had no effect on Bdp1c cross-linking (lane 9) but did increase Bdp1n cross-linking (lane 8). Brf1n-TBPc was able to assemble Bdp1n into the DNA complex (lane 11), but it did not secure cross-linking by Bdp1c (lane 12). Inclusion of Brf1c in the reaction with Brf1n-TBPc enabled the assembly of Bdp1c (lane 14). Brf1n-TBPc was also competent in assembling Bdp1c when both Bdp1n and Bdp1c were present (lane 10), indicating the existence of a binding interface between Bdp1n and Bdp1c and/or the unmasking of a Bdp1c binding domain in Brf1n upon binding Bdp1n. We conclude that Brf1n primarily interacts with the N-terminal half of Bdp1 and that Brf1c primarily interacts with the C-terminal half of Bdp1.


Figure 5
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  		FIGURE 5.
Transcriptional activity of TBPc-Brf1c cluster variants. Multiple-round transcription of the SNR6-derived DNA fragment was performed with 10 nM each Bdp1 and Brf1(1–382) and 20 nM of the TBPc-Brf1c variant identified below the x axis. The mean of at least three experiments normalized to transcription with reference type TBPc-Brf1c is shown (with S.E. values that exceed the symbol size indicated).

 


Figure 6
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  		FIGURE 6.
Brf1c interacts with the SANT domain of Bdp1. A, Brf1(1–382) (Brf1n) and Brf1c interact with the N-(Bdp1n) and C-terminal (Bdp1c) halves, respectively, of Bdp1 split at amino acid 352. Protein-DNA cross-linking with the Brf1(1–382)-TBPc-Brf1(439–596) triple fusion (Brf1n-TBPc-Brf1c) (lanes 2–4), TBPc-Brf1c (lanes 5–9), Brf1n-TBPc (lanes 10–14), or TBPc (lane 1) at 20 nM, and with Bdp1n (N), Bdp1c (C), or both, as indicated above each lane, at 20 nM (lanes 1 and 5–9) or 10 nM (lanes 2–4 and 10–14). TBPc-Brf1c was complemented with 20 nM Brf1n for lanes 8 and 9, and Brf1n-TBPc was complemented with 20 nM Brf1c for lanes 13 and 14. Cross-linked proteins are identified at the sides. B, a Bdp1 segment that is required for binding to Brf1c. Cross-linking reactions were performed with 10 nM wild type Bdp1 or internal deletion variants identified above each lane and 20 nM Brf1n-TBPc (upper panel) or TBPc-Brf1c (lower panel). C, the N-terminal 262 amino acids of Bdp1 do not detectably affect interaction with Brf1c. Protein-DNA cross-linking with 20 nM full-length Brf1 (wild type), 20 nM TBPc or TBPc-Brf1c, and 8 nM full-length Bdp1 or Bdp1(263–594), as indicated above each lane, is shown. D, Bdp1 C-terminally truncated to amino acid 477 is defective in Brf1c interaction. Protein-DNA cross-linking with 20 nM Brf1, 20 nM TBPc (upper panel) or TBPc-Brf1c, and 10 nM Bdp1(138–594) or its variants C-terminally truncated to amino acid 506, 493, 477, or 465, as indicated above each lane, is shown. Cross-linked proteins are identified at the left. (The products marked with asterisks are proteolytic products of Bdp1.).

 
A collection of internal deletions as well as a series of N- and C-terminal truncations of Bdp1 were used to further define the region of Bdp1 that interacts with Brf1c. Bdp1 variants with 12–22-amino acid deletions between Bdp1 residues 253 and 449 were examined for the ability to be recruited to the DNA complex by Brf1n-TBPc or TBPc-Brf1c (Fig. 6B). No deletion within this segment of Bdp1 prevented assembly by Brf1n-TBPc (Fig. 6B, top). In contrast, three deletions covering Bdp1 residues 409–449 effectively eliminated binding to the TBPc-Brf1c·DNA complex (Fig. 6B, bottom). As expected from the ability of TBPc-Brf1c to recruit the C-terminal half of Bdp1 (Fig. 6A), Bdp1(263–594) was also assembled into a DNA complex by TBPc-Brf1c (Fig. 6C). In contrast, Bdp1 C-terminally truncated to amino acid 465 was not recruited by TBPc-Brf1c, and the ability of TBPc-Brf1c to recruit Bdp1 truncated to amino acid 477 was significantly impaired (Fig. 6D, bottom; compare with Bdp1 assembly with wild type Brf1 in the top panel). The ability of TBPc-Brf1c to assemble Bdp1 C-terminally truncated to amino acid 493 or 506 into the DNA complex was substantially retained. These results specify that the entire segment of Bdp1 spanning amino acids 410–476 is essential for Brf1c binding, with an additional contribution provided by amino acids 477–492. This region largely overlaps the SANT domain (amino acid residues 415–472) (27) that is the most highly conserved Bdp1 sequence motif.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Bdp1 is essential for transcription by pol III in vitro, but no single region of Bdp1 is required for TFIIIC-independent transcription of SNR6 as supercoiled DNA (13). Much of Bdp1 can be deleted with retention of yeast viability (the N-terminal 240 amino acids, the C-terminal 108 amino acids, 68 amino acids between residues 312 and 372, and 17 amino acids between 253 and 269), leaving just 161 Bdp1 residues that are essential (26, 28). Nevertheless, there are multiple sites of interaction between Bdp1 and the Brf1·TBP·DNA complex that independently suffice for TFIIIB-DNA complex formation and transcription, and this has been an impediment to identifying the individual sites within Brf1 that are involved in Bdp1 interaction. This work presents the first analysis and identification of Brf1 residues that are involved in assembling Bdp1 into the TFIIIB-DNA complex.

Structure-informed mutagenesis of Brf1(441–506) identifies two separated segments that are involved in Bdp1 assembly. Brf1 residues Asn441,His443, and Leu444 lie above the convex surface of the C-terminal lobe of TBP (Fig. 4B). This region of Brf1 is anchored to TBP by weak van der Waals interaction of Brf1 residues Arg440 and Leu445 with TBP residues Glu228 and Tyr224, respectively. Since this weakly anchored region lies adjacent to the (GS)6 linker, we considered the possibility that the observed defect of Bdp1 assembly by Brf1c N441A/H443A/L444A might be due to an indirect effect, altering the path of Brf1 on the convex surface in a way that would eliminate three polar contacts mediated by the deep intrusion of the Brf1 helix H21 residue Thr448 between TBP helices H2 and H2' (Fig. 1A) (7). The evidence indicates that this is not the case: 1) the single mutations within this cluster were also defective in Bdp1 assembly; 2) with the exception of Pro446 and Ala472, all Brf1 side chains not involved in TBP interaction on the convex surface of TBP were mutated, and none displayed as severe a defect in Bdp1 assembly (Table 1). The amino acid sequence context of this Bdp1-binding epitope, PRNLHLLP, is only conserved within the Ascomycota subphylum Saccharomycotina, and divergence is observed even within this subphylum among the species Candida, Debaryomyces, Pichia, and Clavispora (consensus: PRNLVKNLP). One possible implication of the sequence divergence in this region is that the selective significance that is provided by this site of Bdp1·Brf1 interaction and therefore its overall importance is not dominant in the context of the full-length proteins.

The second Bdp1-binding segment, defined by the deleterious effect of substitutions at Brf1 residues Leu482, Arg485, Ile486, Ile488, and Leu490, lies at the outward facing, exposed surface of Brf1 helix H24 at the N-terminal side of TBP (Fig. 4B). The helix H24 region of Brf1 is a major interface with TBP, contributing six intersubunit hydrogen bonds in yeast (four in mammals) (7), and is conserved from yeast to mammals (29–31). However, the residues identified here as important for Bdp1 assembly are also conserved between the anciently diverged (1.0–1.2 Myr (32, 33)) fungal phyla Ascomycota ((I/L)RI(L/V)(I/E)) and Basidiomycota ((I/L)RV(L/V)X) and partly retained in chordate Brf1 ((V/I)ELM(Q/E)) (supplemental Fig. S2). If this Bdp1-Brf1 interface is retained in vertebrate Brf1, it is noteworthy that this part of the Brf1 helix H24 is clearly absent from the Brf1 paralogue Brf2 (34). A prior in vivo analysis (35) of the effect of sequence substitutions in 19 residues of Brf1 between amino acid residues 453 and 508 did not reveal any growth defect that could be ascribed to a defect in interaction with Bdp1. Given that 18 of those substitutions replaced charged amino acids, this negative result is consistent with our analysis: only one charge reversal, R485E, substantially impaired Bdp1 assembly (Table 1). In fact, most of the Brf1 residues identified here as important for Bdp1 assembly contain large hydrophobic side chains (Leu444, Leu482, Ile486, Ile488, Leu490). TFIIIB-DNA complexes are distinctive in that they are salt- and heparin-resistant (12). The interaction of Bdp1 with Brf1 helix H24 may be predominantly hydrophobic, and this may contribute to the stability of TFIIIB-DNA complexes at high salt concentrations.

TBPc-Brf1c variants D449K, E463K, E468K, L495A, K504E, and E506K displayed a more modest (2-fold) defect in Bdp1 assembly. In fact, only three TBPc-Brf1c variants (D458K, E484K, and E497K) assembled Bdp1 with mean cross-linking efficiencies that consistently closely matched or exceeded the reference TBPc-Brf1c. We propose that the Bdp1-binding interface of Brf1c is extensive and that additional sites along the entire segment of Brf1c that was resolved in the structure of Brf1c-TBPc-DNA ternary complex (7) contribute to the affinity of Brf1c for Bdp1c in the TFIIIB·DNA complex. The N-terminal end of Brf1c and Brf1 helix H24 provide the high affinity binding sites for Bdp1, with numerous weaker sites of Bdp1 interaction in between, as well as C-terminal of helix H24.

The segment of Bdp1 that interacts with Brf1(439–596) has been localized by deletion analysis primarily to amino acids 410–476, with Bdp1 residues 477–492 also significantly contributing to Brf1 binding (Fig. 6). The SANT domain (residues 415–472), which is the most highly conserved Bdp1 motif (43% identity between S. cerevisiae and H. sapiens (36)), constitutes a large part of this Bdp1 segment. Although there is a strong similarity between SANT domains and the DNA-binding domain of Myb-related proteins, the involvement of the SANT domain in protein-protein interaction has also been documented (37–39). It is an intriguing possibility that the co-conservation of helix H24 of Brf1s and the SANT domain of Bdp1s results from a conserved interaction between these domains. In this regard, Brf1 residues Leu482, Arg485, Ile486, and Leu490 (and TBP residue Glu93), which are involved in Bdp1 binding face upstream (relative to the direction of transcription). The region of Bdp1 that cross-links to bp -33 on the nontranscribed strand just upstream of the SNR6 TATA box also largely coincides with the SANT domain (between Bdp1 residues 425 and 485) (40). The amino acid 419–455 segment of the Bdp1 SANT domain can be reasonably compared with the structure of the human KIAA 1915 protein's SANT domain (37% identity, 59% similarity; Protein Data Bank entry 2CU7), which forms a compact helix-loop-helix-loop-helix structure; two additional {alpha} helices are predicted within the residue 456–491 segment of Bdp1. This latter segment would need to be extended in order to span the ~60-Å distance between Brf1c helix H24 and the N terminus of Brf1c. It is likely that the loss of Brf1 binding by any deletion that encroaches into the Bdp1 410–492 region results at least in part from a gross alteration of Bdp1 tertiary structure. A more precise specification of residues in the SANT domain region of Bdp1 that are involved in Brf1 interaction will require site-directed mutagenesis and determination of structure.

An additional outcome of our analysis is the finding that most of the binding affinity of Brf1n (Brf1(1–382)) for Bdp1 is contributed by Bdp1n (Bdp1(1–352)) (Fig. 6A). Since the TFIIB-related Brf1(1–282) segment forms a transcriptionally active but unstable TFIIIB-DNA complex with Bdp1 and TBP (6, 19), it is likely that this Bdp1n-binding site resides within Brf1(1–282). TFIIIB-DNA complexes formed with Bdp1 and the Brf1n-TBPc fusion protein are stable to electrophoretic mobility shift assay, and this should aid in fine mapping the Brf1n·Bdp1n interface.

C-terminal truncations of TBPc-Brf1c to amino acids 510 and 497 essentially abolished transcription of a linear DNA, yet had little effect on Bdp1 assembly (Fig. 2). As already noted, this observation is largely in agreement with Ref. 22. However, TBPc-Brf1c cluster mutants K501E/R502D/K504E and E506K/D508K also displayed a severe defect in transcription but at most a modest decrease in Bdp1 assembly (<2-fold decrease with 10 nM Bdp1) (Figs. 3 and 5). Whereas these clustered charge reversal mutations may partly disrupt Brf1 helix H25 or alter a potential interaction of this helix with downstream DNA sequence resulting in a loss of Brf1-pol III interaction elsewhere in the Brf1(510–596) C-terminal segment, it is noteworthy that the cluster mutant E506A/D508A confers a cold-sensitive growth phenotype (mutant II.16 (35)) similar to K594A/K595A (mutant III.2), for which genetic evidence of an interaction with the C34 subunit of pol III has been inferred. It has been proposed that E506A/D508A alters the conformational flexibility of Brf1, since this mutation effectively shields a nearby site from proteolysis during purification (35).


   FOOTNOTES
 
* This work was supported by NIGMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

2 Present address: The Wellcome Trust and Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, United Kingdom. Back

1 To whom correspondence should be addressed: Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0634. Tel.: 858-534-2451; Fax: 858-534-7073; E-mail: gak{at}biomail.ucsd.edu.

3 The abbreviations used are: pol, RNA polymerase; TBPc, TBP(61–240); Brf1c, Brf1(439–596); Brf1n, Brf1(1–382); Bdp1n, Bdp1(1–352); Bdp1c, Bdp1(352–594); TBP, TATA-binding protein. Back


   ACKNOWLEDGMENTS
 
We are grateful to M. Ouhammouch for advice and to M. Ouhammouch and F. Saida for a careful and incisive reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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T. Chung, O. Siol, T. Dingermann, and T. Winckler
Protein Interactions Involved in tRNA Gene-Specific Integration of Dictyostelium discoideum Non-Long Terminal Repeat Retrotransposon TRE5-A
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