Yeast Mitochondrial Transcription Factor Mtf1 Determines the Precision of Promoter-Directed Initiation of RNA Polymerase Rpo41

Abstract

Despite their clear T7-bacteriophage origin, mitochondrial RNA polymerases have evolved to require transcription factors. All mitochondrial polymerases contain an extra N-terminal domain that has no counterpart in the self-proficient phage enzyme, which is therefore hypothesized to interact with transcription factors. We studied a series of N-terminal deletion mutants of yeast mitochondrial RNA polymerase, Rpo41, and have found that the N-terminal region does not abolish the effects of Mtf1; rather it contributes directly to enzyme catalysis. Mtf1 can rescue the defective Rpo41 enzymes resulted from N-terminal domain deletions. Although Rpo41 appears to have retained all promoter recognition elements found in T7 RNAP, the elements are not independently functional, and Mtf1 is necessary and sufficient for holoenzyme promoter-directed transcription activity.

Fig 1. Sequence alignment and structural model.

(A). a T7 RNAP initiation complex showing the 17 bp promoter (purple) is recognized by AT-rich minor groove recognition loop, the intercalating hairpin and the specificity loop (all colored green). The NTD (yellow) and CTD (blue) are indicated. (B). Primary sequence and secondary structure of the NTD of Rpo41 indicating the deletion sites. A sequence alignment of T7 RNAP with the human and yeast mitochondrial RNAPs is presented in S1 Fig (C). Purities of the Mtf1 and Rpo41 variants analyzed by SDS-PAGE.

Fig 2. Activities of Rpo41 variants on a linear template.

Transcription of 1 μM 70-bp linear DNA containing 14S rRNA (A) using 0.5 μM Rpo41 (wild-type or a variant) without or with 2.5 μM Mtf1 were performed in the presence of NTPs and [γ-³²P]ATP (B). (C). The intensities of 34- mer transcripts in (B) were calculated by Quantity One software (BioRad).

Fig 3. Activities of Rpo41 variants on a supercoiled DNA containing a 14S rRNA promoter.

(A). Transcription was carried out in the presence of ATP, UTP, GTP and [γ-³²P]ATP (no CTP) with 0.5 μM Rpo41 (wild-type or a variant) in the absence or presence of 0.5 μM Mtf1 using 0.2μM pJJ1305 as template. (B). The 107-mer RNA products quantified using software. Quantity One.

Fig 4. Interactions of Rpo41 with Mtf1 and promoter DNA.

(A). Analytical gel filtration chromatograph of wild-type Rpo41 (2.8 μM) with Mtf1 (5.6 μM) (red) superimposed with that of Rpo41 (1 μM), Mtf1 (2 μM) and a 20bp promoter-containing DNA duplex (3 μM) (blue). The elution volume of Rpo41 alone is indicated (black arrow). The protein contents of the peak fractions of Rpo41 + Mtf1 and Rpo41 + Mtf1 + DNA were visualized after SDS-PAGE. (B). Analyses described in (A) performed with Rpo41 ΔN393.

Fig 5. Activities of Rpo41 variants on pre-melted promoter-containing DNA.

(A). The duplex DNA template contains a 14S rRNA promoter, with non-complementary sequences from positions-4 to +2. (B) (1) [γ-³²P]ATP labeled transcripts on the above template (1 μM) using Rpo41 (0.5 μM wild-type or a variant) in the absence or presence of Mtf1 (2.5 μM) were visualized on a polyacrylamide denaturing gel. (2) Same as (1), except that transcripts are labeled with [γ-³²P]ATP, [γ-³²P]GTP or [α-³²P]ATP. C) The intensities of run-off transcripts in B (1) quantified using Quantity One software.

Fig 6. Comparisons of promoter strength and structure.

(A). Sequences and structures of the 65-bp constructs contain a linear promoter and a pre-melted region with or without 14S rRNA promoter. (B-D). transcripts yielded from the ab0ove constructs using Rpo41 wild-type or ΔN160 (0.5 μM) in the absence or presence of Mtf1 (2.5 μM), labeled with [γ-³²P]GTP, [γ-³²P]ATP, and [α-³²P]ATP, respectively.

Fig 7. Summary of Rpo41 and Mtf1 roles in RNA transcription.

The Rpo41 exhibits promoter-specific activity on closed or open template in the presence of Mtf1 (black pathway). The holoenzyme containing Rpo41 ΔN1 deletion mutant presents normal activity. However, Rpo41 alone exhibits only non-specific activity on both closed and open promoter (red). Rpo41 ΔN1 deletion mutations alone are defective in transcription.