Identifying a core RNA polymerase surface critical for interactions with a sigma-like specificity factor

Abstract

Cyclic interactions occurring between a core RNA polymerase (RNAP) and its initiation factors are critical for transcription initiation, but little is known about subunit interaction. In this work we have identified regions of the single-subunit yeast mitochondrial RNAP (Rpo41p) important for interaction with its sigma-like specificity factor (Mtf1p). Previously we found that the whole folded structure of both polypeptides as well as specific amino acids in at least three regions of Mtf1p are required for interaction. In this work we started with an interaction-defective point mutant in Mtf1p (V135A) and used a two-hybrid selection to isolate suppressing mutations in the core polymerase. We identified suppressors in three separate regions of the RNAP which, when modeled on the structure of the closely related phage T7 RNAP, appear to lie on one surface of the protein. Additional point mutations and biochemical assays were used to confirm the importance of each region for Rpo41p-Mtf1p interactions. Remarkably, two of the three suppressors are found in regions required by T7 RNAP for DNA sequence recognition and promoter melting. Although these essential regions of the phage RNAP are poorly conserved with the mitochondrial RNAPs, they are conserved among the mitochondrial enzymes. The organellar RNAPs appear to use this surface in an alternative way for interactions with their separate sigma-like specificity factor, which, like its bacterial counterpart, provides promoter recognition and DNA melting functions to the holoenzyme.

FIG. 1

Features of Mtf1p and Rpo41p. (A) Comparison of Mtf1p to sigma factors and location of Mtf1p mutations that affect interactions with Rpo41p. Mtf1p mutant V135A is highlighted. Regions with sequence similarity to sigma factors are shaded (24). aas, amino acids. (B) Comparison of Rpo41p to T7 RNAP. Regions with high levels of identity are shaded. Locations of mutations in Rpo41p suppressors I30, I12, and V1 and convenient restriction enzyme sites used for subcloning the mutations are indicated. Fragments capable of converting the wild-type sequence to a suppressor are depicted as thicker lines. Mutations responsible for suppression are in boldface.

FIG. 2

Three Rpo41p constructs suppress the core-binding defect of Mtf1p mutant V135A. β-Galactosidase activity is shown for wild-type (WT) Rpo41p and the suppressor constructs interacting with Mtf1p mutant V135A. The wild-type Rpo41p interaction with wild-type Mtf1p is shown as a reference. The activity is shown for cells grown at 23°C. β-Galactosidase activity is expressed as Miller units (34).

FIG. 3

Interaction of V135A suppressors with wild-type (WT) Mtf1p (A) and Mtf1p mutant K157E (B). Cells were grown at 30°C. β-Galactosidase activity is expressed as Miller units (34).

FIG. 4

Biochemical confirmation of interactions between the suppressors and Mtf1p mutant V135A. Whole-cell yeast extracts were prepared as described in Materials and Methods from cells expressing only wild-type (WT) or mutant forms of Rpo41p. The extracts were passed over a column of purified GST-Mtf1p that contained the V135A mutation. The columns were washed to eliminate nonspecific binding and then step eluted to release Rpo41p bound to the column. Rpo41p from input (IP), flowthrough (FT), wash, and elution fractions was detected by Western blot analysis using anti-Rpo41p antibody. The arrowheads on the left indicate a cross-reacting band (see also Fig. 7).

FIG. 5

Structural and amino acid sequence features near the RPO41 suppressor mutations. Each panel includes a schematic of structural elements flanking a suppressor mutation (position indicated by the asterisk). Cylinders and arrows denote helices and beta sheets, respectively, from the structure of T7 RNAP (25). Below each schematic is an amino acid alignment comparing the core RNAP from S. cerevisiae (Sc; accession no. M17539) to sequences from N. crassa (Nc; L25087), S. pombe (Sp; P13433), and Homo sapiens (Hs; 4826926). The boxed amino acids indicate the original suppressor; boldface indicates positions and identities of site-directed mutations described in the text. (A and B) The consensus below the mitochondrial RNAP alignments uses an uppercase letter for a four-of-four match, lowercase for a three-of-four match, and + for similar amino acids. Below the mitochondrial RNAPs is the relevant sequence from T7 RNAP (M38308) and a consensus derived from this sequence and those of phage T3 (X02981), K11 (X53238), and SP6 (Y00105). Highlighted in boldface are T7 RNAP positions involved in promoter melting or stabilization of single-stranded DNA (filled circles) and amino acids important for promoter recognition (arrowheads) as described in the text. (C) The consensus for the two fungal sequences uses uppercase for identity and + for similarity.

FIG. 6

Analysis of site-directed Rpo41p mutations by two-hybrid and plasmid shuffle assays. Mutations were cloned into two-hybrid and yeast expression vectors as described in Materials and Methods. Two-hybrid interactions were measured for cells grown at 30°C; β-galactosidase activity is expressed as Miller units (34). The ability of the mutations to sustain growth on a nonfermentable carbon source (glycerol-ethanol-lactate) is indicated in the boxes below the bar graph. WT, wild type.

FIG. 7

Core RNAP activity of noninteracting Rpo41p mutants. As described in Materials and Methods, transcription extracts were made from yeast cells expressing wild-type (WT) Rpo41p or the indicated Rpo41p mutants. (A) Western blot of the transcription extracts with anti-Rpo41p antibody. The arrow on the left indicates the position of Rpo41p; the arrowhead on the right indicates a cross-reacting band. (B) The extracts were passed over a DEAE-cellulose column to remove nuclear RNAPs. The adsorbed fractions were assayed to determine relative amounts of mitochondrial RNAP, using poly(d[AT]) as a nonspecific template.

FIG. 8

Rpo41p suppressor mutations modeled on the T7 RNAP structure. Coordinates are from reference . The domain colors are similar to the scheme of Jeruzalmi and Steitz (25): N-terminal domain, yellow; thumb, green; palm, red; palm insertion, orange; fingers, blue; pinky specificity loop, light blue; and extended foot, magenta. The approximate locations of the suppressor mutations (as predicted by the alignments shown in Fig. 5) are shown as starred circles on the RNAP structure and are indicated by arrows.