Figure S1: rtf1 cDNA and encoded protein sequence. Arrows indicate the positions of the
identified six introns. rtf1 mutations and corresponding substitutions are given. Twenty rtf1 mutant
alleles were identified using polymerase chain and sequencing reactions. Of these, two mutant
alleles were isolated independently twice, while one mutation was isolated three times in the same
batch of UV mutagenesis.
Figure S2: A) SDS-Page gels of purified the Rtf1 domains. The molecular sizes of marker
proteins are given to the left. B) Western analysis of shifted material. Gel shifts were obtained in
duplicate under standard conditions (domains and linkers are given in the panel). One set of gelshifted materials was used directly for obtaining an autoradiogram (three left panels marked gel
shift), while the other was used for a Western analysis using an -MBP antibody for detection of
the MBP-Rtf-domain fusion proteins (three panel marked Western). The analysis detects the fusion
proteins at the same position in the gels as the shifted substrate. C) Domain I binding to reb4 and
regA dsDNA, and domain II binding to ls-regA ssDNA can be out-competed by addition of a
specific competitor. Gel-shift assays were performed under standard conditions in the presence of
the unspecific competitor poly G:C. However, after the 30 min. incubation with the labeledsubstrate, 10 fold excess unlabeled substrate was added and the sample incubated for additional 30
min., followed by gel analysis. D) 2D-gel analysis of replication intermediates from the rtf1-L162Y
strain. The PstI-SacI fragment of plasmid pSC11 is analyzed. An analysis of plasmid pSC11 in the
wild-type background is shown in Fig. 5C lower panel. E) The RTS1 sequence is required barrier
activity. 2D-gel analysis of the empty plasmid pRep3 in mutant rtf1-S154L. An analysis of plasmid
pBZ142 containing the full length RTS1 element in mutant rtf1-S154L is shown in Fig. 5F. F) 2Dgel analysis of plasmid pSC11 in the swi1, rtf1-S154L and swi3, rtf1-S154L double mutant
strains (JE178, JE180). The Rtf1-S154L barrier signal (blue arrows) is absent in these strains. Note
that the apex of the arcs in these gels appears more intense, however, this is not due to barrier
activity but due to that the width of the arc is decreased (also see Fig. 5A in Codlin & Dalgaard,
2003). G) Polymerase II transcription from the nmt1 promoter does not affect replication
termination at the wild-type RTS1. 2D-gel analysis of plasmids pBZ142 (panel H) and pBZ143
(inverted RTS1) in the absence and presence of transcription. The repressed state was achieved by
addition of 10 mM thiamine (Maundrell, K., 1993). H) Graphic outline of the plasmid pBZ142.
The position and orientation relative to the plasmids nmt1 promoter and replication origin are
given. I) Weak residual barrier activity with an inverted polarity is observed at the genomic RTS1
locus in the rtf1-S154L genetic background. 2D-gel analysis of the genomic NsiI fragment. J) The
rtf1-R346* mutation abolish RTS1 barrier activity. 2D-gel analysis of RTS1 replication
intermediates. The PstI-SacI fragment of plasmid pSC11 is analyzed.
S. pombe strains:
JE148, h90, ade6-216, leu1-32, rtf1-S154L;
JE178, RTS1-mat1-SspI::RTS1, leu1-32, ura4-D18, ade6-210, swi1::ura4+, pSC11;
JE180, RTS1-mat1-SspI::RTS1, leu1-32, ura4-D18, ade6-210, swi3-146, pSC11;
JZ1, h90, ade6-210, leu1-32, ura4-D18 (Dalgaard and Klar, 1999);
JZ183, RTS1-mat1-SspI::RTS1, leu1-32, ura4-D18, ade6-210 (Dalgaard and Klar, 2000);
JZ264, RTS1-mat1-SspI::RTS1, leu1-32, ura4-D18, ade6-216;
SC8, RTS1-mat1-SspI::RTS1, leu1-32, ura4-D18, ade6-216, rtf1::ura4+(Codlin and Dalgaard, 2003);
SC46, h90, leu1-32, ura4-D18, ade6-216, rtf1::ura4, pBZ142 (Codlin and Dalgaard, 2003);
Plasmids:
pBZ129, pHW5 containing a 8.8 Kb genomic rtf1+ HindIII fragment;
pBZ136, pHW5 containing a 3.8 Kb genomic rtf1+ HindIII fragment;
pBZ142, pREB3 (Maundrell, 1993) containing RTS1 (orientation 1; Codlin and Dalgaard, 2003);
pBZ143, pREB3 (Maundrell, 1993) containing RTS1 (orientation 2; Codlin and Dalgaard, 2003);
pSC11, pREB3 containing region B (Codlin and Dalgaard, 2003);
pSC4, pREB3 containing the minimal RTS1 element (Codlin and Dalgaard, 2003);
S. cerevisiae strain:
AH109, MATa, trp1-901, leu2-3, ura3-52, his3-200, gal4, gal80, LYS2::GAL1UAS-GAL1TATAHIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1TATA-lacZ. (BD Biosciences Clontech)
Identification of the rtf1 gene. The rtf1 gene was cloned by complementation of the iodinestaining phenotype of rtf1 mutant strain JZ231 using a HindIII genomic library. One transformant
containing a complementing plasmid was identified. The plasmid (pBZ129) was isolated and
complementation was confirmed by retransformation. Subsequently, a linkage analysis was
performed to verify that it was the rtf1 gene that had been cloned and not a multi-copy suppressor.
Plasmid pBZ129 was integrated into the genome of the rtf1 mutant strain (JZ202) by homologous
recombination. The obtained strain, JZ316, was backcrossed to the parental strain (JZ183). Tetrad
analysis of asci obtained from the cross detected no crossovers between the rtf1 mutation and the
S. cerevisiae LEU2 marker present on the integrated plasmid (18 tetrads analyzed; data not
shown). Sequence analysis revealed that pBZ129 contains an 8.8 Kb genomic fragment.
Subsequently, sub-cloning and complementation studies identified rtf1 as the open reading frame
SPAC22F8.07C defined in the S. pombe genome project. The open reading frame was determined
by RT-PCR from isolated total RNA followed by sequence analysis. The comparison of genomic
and cDNA sequences identified the positions of six introns (supplementary figure 1). Database
searches revealed that Rtf1 is a member of the transcription termination factor family that includes
Reb1 from S. pombe (32% identity in amino acids, AAs, region 97 to 421), S. cerevisiae and
Kluyveromyces lactis, and the TTF1 factors from mouse and human (Fig. 2B).
Computational analysis of the relationship between the c-myb and the Rtf1/TTF1/Reb1
protein family. We took three different approaches: First, the PSI-BLAST alignments and eight
proteins (Figure 2B; SpRtf1, SpReb1, SpEta2, ScReb1, ScReb1L, HsTTF1, HsDMTF1, Mmcmyb_1H89) were used to estimate a hidden Markov model (HMM) of the two ~200 AA Rtf1/Reb1
conserved segments. This analysis was done using the Sequence Alignment and Modeling
Software System v3.5 (Hughey and Krogh, 1996; Krogh et al., 1994). Subsequently, the
Rtf1/Reb1/c-myb HMM was used to generate a multiple sequence alignment of the eight training
sequences (Fig. 2B). The alignment illustrates a low but significant sequence homology between
the repeated domains. Second, to define the boundaries of myb structural motifs, the Mmcmyb_1H89 amino acid sequence and PSI-BLAST were utilized to search the Conserved Domains
Database (Marchler-Bauer et al., 2005). Each myb motif (Myb1, Myb2, Myb3) is a member of
Pfam family “Myb_DNA-binding” (pfam 00249.12) that contains the DNA binding domains from
Myb proteins, as well as the SANT domain family. Importantly, this analysis suggested the
presence of three myb-like structural motifs in each of the two domains, each consisting of three
alpha helices, suggesting that for this protein family up to six different sub-domains might interact
with the DNA recognition site. Third, due to the low although significant sequence similarity (PSIBLAST score E<0.05) for domain I, we made an independent prediction of the secondary
structural motifs of the Rtf1 domain I sequence using the PHD program (Rost et al., 2004).
Notably, the secondary structural motifs predicted by this program for Rtf1 domain I correspond to
the alpha-helices observed for the c-myb structure and align accordingly (Figure 2B).
Protein expression, purification and gel-shift assays: Domain I was cloned into the pMAL-c2x
vector (New England Biolabs), and obtained plasmids were transformed into E. coli strain TB1 (Fara(lac-proAB)[80dlac(lacZ)M15] rpsL(StrR) thi hsdR; Clontech). Partial purification was
done using an amylose column (di Guan et al., 1988). The domain II was expressed using plasmid
pIVEX (Roche) and E. coli strain BL21(DE3) (Studier and Moffatt, 1986). Expression was
maintained for 2 hours before harvest. Partial purification was obtained using a Ni2+ column
(Petty, 1996) followed by an amylose column. Domain I+II was expressed using using plasmid
pIVEX (Roche) and E. coli strain BL21/phage CE6 (Studier and Moffatt, 1986). Inclusion
bodies were isolated, the protein dissolved in 6 M Urea. Subsequently, the protein was renatured
by binding a nickel column and slow removal of the Urea, and finally eluted using imidazole. The
final protein samples’ concentrations were determined using the Bradford assay (Bradford, 1976).
Gelshifts were obtained as described by Sambrook and Russell, 2001. 1 x binding buffer contained
20 mM Tris-HCl pH 7.5, 1 mM MgCl2, 0.5 mM EDTA and 0.5 mM -mercaptoethanol. In
addition, 4% glycerol or 0.5 mg/ml bovine serum albumin was added for domain I and domain II
C-terminus, respectively. Experimental conditions were as follows: 150 pmol/ml P32-endlabled
oligonucleotides; 100 g/ml nonspecific competitor unless otherwise indicated; 0.5 x binding
buffer, except for figure 3A & B were 1 x binding buffer was employed; Purified protein
concentrations as listed for each figure: 3A & B, 83 g/ml; 3C, 167 or 334 g/ml; 3D, domain I
167 or 334 g/ml, domain II 275 or 545 g/ml; 3E, 54 g/ml; 3F, domain II 54 g/ml, domain II
W405Gg/ml; 4A, domain I 83 g/ml, domain I+II 54 g/ml; 4B, both 54 g/ml; 4C, 109
g/ml; 5F, both 20 or 60 g/ml; S2, domain I 83 g/ml, domain II 109 g/ml, domain I+II 54
g/ml. Samples were incubated for 30 min. on ice followed by an analysis on a 5% 37.5/1
acrylamide/bisarcylamide gels, which were run at 100V for 3 hours at 4C in 0.5 x TBE buffer. In
competition assays the domains were first incubated in the presence of one substrate on ice for 30
min on ice, the other was then added and the incubation continued for 30 min at room temperature.
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