Figure 1. S. cerevisiae and human mtRNAPs cap RNA with NAD⁺ and NADH in vitro.

(A) Structures of ATP, NAD⁺, and NADH. Grey, identical atoms; black, distinct atoms. (B) Processing of RNA 5' ends by RppH and NudC. A, adenosine; N⁺, NAD⁺ nicotinamide; N, NADH nicotinamide; p, phosphate. (C and D) NCIN capping with NAD⁺ and NADH by S. cerevisiae mtRNAP (C) and human mtRNAP (D). Top, promoter derivatives. Middle, initial RNA products of in vitro transcription reactions with ATP, NAD⁺, or NADH as initiating nucleotide and [α³²P]-GTP as extending nucleotide. Bottom, full-length RNA products of in vitro transcription reactions with ATP, NAD⁺, or NADH as initiating nucleotide and [α³²P]-GTP, ATP, UTP, and 3'-deoxy-CTP (C), or [α³²P]-GTP, ATP, and UTP (D) as extending nucleotides. Products were treated with RppH or NudC as indicated. Grey box and arrow, transcription start site (TSS);+31 and+17/18, position of last NTP incorporated into full-length RNA products; M, 10-nt marker.

Figure 1—figure supplement 1. S. cerevisiae and human mtRNAPs cap RNA with NAD⁺ in vitro: additional data.

(A) Processing of radiolabeled RNA 5'-ends by alkaline phosphatase (AP) and NudC. A, adenosine; N⁺, NAD⁺ nicotinamide; p, phosphate. *, radiolabeled phosphate. (B and C). NCIN capping with NAD⁺ by S. cerevisiae mtRNAP (B) and human mtRNAP (C). Top, promoter derivatives. Bottom, full-length RNA products of in vitro transcription reactions with [γ³²P]-ATP or [α³²P]-NAD⁺ as initiating nucleotide and GTP, ATP, UTP, and 3'-deoxy-CTP (C), or GTP, ATP, and UTP (D) as extending nucleotides. Products were treated with NudC alone, AP alone, or NudC and AP, as indicated. Grey box and arrow, TSS; +31 and +17/18, position of last NTP incorporated into RNA; M, 10-nt marker.