The transcription factor Spn1 regulates gene expression via a highly conserved novel structural motif

Abstract

Spn1/Iws1 plays essential roles in the regulation of gene expression by RNA polymerase II (RNAPII), and it is highly conserved in organisms ranging from yeast to humans. Spn1 physically and/or genetically interacts with RNAPII, TBP (TATA-binding protein), TFIIS (transcription factor IIS), and a number of chromatin remodeling factors (Swi/Snf and Spt6). The central domain of Spn1 (residues 141-305 out of 410) is necessary and sufficient for performing the essential functions of SPN1 in yeast cells. Here, we report the high-resolution (1.85 Å) crystal structure of the conserved central domain of Saccharomyces cerevisiae Spn1. The central domain is composed of eight α-helices in a right-handed superhelical arrangement and exhibits structural similarity to domain I of TFIIS. A unique structural feature of Spn1 is a highly conserved loop, which defines one side of a pronounced cavity. The loop and the other residues forming the cavity are highly conserved at the amino acid level among all Spn1 family members, suggesting that this is a signature motif for Spn1 orthologs. The locations and the molecular characterization of temperature-sensitive mutations in Spn1 indicate that the cavity is a key attribute of Spn1 that is critical for its regulatory functions during RNAPII-mediated transcriptional activity.

Figure 1

Temperature-sensitivity of spn1 mutants is not due to instability of the protein at the restrictive condition. (a) Yeast strains as indicated were serially diluted, plated onto YPD plates and incubated at 30°C or 38°C for two or four days, respectively. (b) Yeast cells containing both untagged wild-type Spn1 and myc-tagged wild-type, myc-tagged Spn1-K192N, myc-tagged Spn1-D172G or myc-tagged Spn1-L218P were grown to mid-log phase and then incubated an additional hour at 30°C or 38°C prior to harvesting. Protein extracts were prepared from the cells and Spn1 levels in the extracts evaluated by immunoblot analysis with polyclonal anti-Spn1 antibodies.

Figure 2

Mutations in SPN1 reduce the Spn1-RNAPII and Spn1-Spt6 interactions and confer changes in the regulation of CYC1 transcription. (a) Protein extracts prepared from the indicated strains were immunoprecipitated with protein A-Sepharose beads coupled to polyclonal anti-Spn1 antibodies. RNAPII was detected by immunoblot analysis with antibodies to the largest subunit of RNAPII. The load (L) represents 5% of the input material for the IP of which 50% was loaded. (b) Protein extracts prepared from the indicated SPN1 strains also containing an HA-tagged version of Spt6 were immunoprecipitated with protein A-Sepharose beads coupled to anti-HA antibodies. Spn1 derivatives were detected by immunoblot analysis with polyclonal anti-Spn1 antibodies. The load and IP are as in panel a. (c) The effect of spn1-K192N, spn1-D172G and spn1-L218P on CYC1 transcription was evaluated in an S1 Nuclease protection assay. The indicated strains were grown to mid-log phase in medium containing dextrose (CYC1 partial repression) and then transferred to medium with ethanol (CYC1 activation) for the times indicated (in hours). Total cellular RNA was isolated, hybridized to radioactive CYC1 and tRNA^W probes, digested with S1 Nuclease and the products separated on denaturing polyacrylamide gels. Relevant regions of a representative gel are shown. The tRNA^W probe was included as a load control and was used to normalize CYC1 expression.

Figure 3

The structure of the conserved central domain of yeast Spn1. (a) primary sequence alignment of Spn1 from Saccharomyces cerevisiae (Sc) and Homo sapiens (Hs) is shown. Identical amino acids are underlined and shown in dark green with similar amino acids in light green. Numbers indicating the starting and ending amino acid positions are shown at the left and right, respectively for each homolog. Secondary structure elements are indicated above the primary amino acid sequence. (b) Overall structure of the highly conserved central domain of yeast Spn1 shown in ribbon format (left) and space-filling mode (right), with the cavity indicated (arrow). Mini-Spn1 consists of eight α-helices (α1 to α8) and four loops (L1 to L4), and contains residues from 149 to 293. From top to bottom, the asterisks denote the locations of L176, L228 and I238 respectively (see text for details).

Figure 4

Four hydrogen bonds stabilize the L2 loop in chain A. (a) E226 forms a hydrogen bond with the side chain of R222. (b) The L225 carbonyl group forms a hydrogen bond with the Y268 hydroxyl group. (c) The P234 carbonyl group forms a hydrogen bond with the Q239 side chain. (d) The D230 carbonyl oxygen forms a hydrogen bond with the R273 side chain. In panels a, b, and d the mini-Spn1 structure has been rotated 180 degrees from the orientation in Figure 3B. To optimize visualization of the hydrogen bonds, panels b and d have been rotated slightly into the plane of the paper.

Figure 5

The surface of mini-Spn1 has a number of conserved and distinctive structural aspects. (a) The sequence conservation as determined from the alignment of Spn1 from Saccharomyces cerevisiae and Homo sapiens is plotted on the front (left) and back (right) sides of Spn1. The top panel shows the structure in a ribbon diagram. The lower panel shows the structure in a space-filling model. Dark green indicates identical residues and light green similar residues. (b) The electrostatic potential of mini-Spn1 is shown on its molecular surface. The molecule is shown in the same orientation as in (a). The basic (blue) and acidic (red) residues are indicated. (c) Hydrophobic residues are shown (orange) on the surface of mini-Spn1. The cavity region of Spn1 is rimmed with hydrophobic amino acids as can be seen on both the front- and back sides. The front surface of mini-Spn1 also has a pronounced region comprised of hydrophobic residues that extends downward from the cavity.

Figure 6

The location of D172, K192 and L218 on the mini-Spn1 structure is shown. (a) D172 is located on helix α1 while K 192 is on helix α2. (b) L218 is located at the beginning of helix α4 within the hydrophobic core region. The mini-Spn1 structure shown in Panel B has been rotated 180 degrees relative to Panel a.

Figure 7

Spn1 and mouse TFIIS protein 3 have primary sequence and structural similarities. (a) The yeast mini-Spn1 and mouse TFIIS (PDB ID: 1WJT) protein structures are shown in ribbon format. The structural similarity begins with helix α4 of Spn1 and helix α1 of TFIIS. (b) The primary sequence alignment of Spn1 (residues 213-291) and mouse TFIIS (residues 1-77) is shown above with conserved amino acids underlined and shown in dark green. Similar residues are shown in light green. Amino acid insertions are indicated with a period in opposite sequence. The secondary structure of mini-Spn1 is shown above the sequence alignment. The secondary structure of domain I of mouse TFIIS protein 3 is shown below the sequence alignment.