Transcription Initiation on DNA Templates in Cell-Free Systems

Inhibition of Transcription Initiation on DNA Templates in Cell-Free Systems 

The availability of procedures to isolate mammalian DNA-de-pendent RNA polymerases and their cofactors in order to reconstitute them with DNA templates for in vitro initiation of transcription provided an essential avenue for investigating VSV inhibitors of RNA synthesis. McGowan et al. (1982) exploited the in vitro transcription system developed by Manley et al. (1980) to test the capacity of the 

This system provided the first opportunity to test whether the VSV wt leader is the viral product responsible for the inhibition of the initiation of DNA-dependent RNA synthesis. Later studies (McGowan and Wagner, unpublished data) indicate that, when DNA templates transcribed by RNA polymerase II and III in vitro are used simultaneously in the same reaction, polymerase II activity is inhibited more easily than RNA polymerase III transcripts, confirming previous in vivo data obtained by Week and Wagner (1978, 1979 ). 

McGowan et al. (1982) compared the known nucleotide sequence of plus-strand wt leader with that of the DI leader (Colonno and Ba-neijee, 1978a Schubert et al., 1978), in an attempt to identify sequences unique to the wt leader that could be involved in transcription inhibition. As shown in Fig. 2, the wild-type leader RNA of VSV-Indiana has at its 5 end one region (nucleotides 1-17) nearly identical to that of the 5 end of the DI leader this region is conserved both at the 3 and 5 ends of all strains of VSV wild-type and DI particles (Schubert et al., 1978) and may serve as a nucleation signal for en- 

Although the case for inhibition of DNA-dependent RNA synthesis by wt VSV leader RNA sequences is building, all that can really be said at this time is that wt VSV leader RNA contains nucleotide sequences potentially capable of interacting with promoters or with host cell protein cofactors that interact with nucleotide sequences essential for accurate transcription. Perhaps the most intriguing possibility is that VSV wt leader RNA can serve as a surrogate for other small RNA species found inside the cytoplasm and nucleus of cells (Lerner and Steitz, 1981 Lerner et ah, 1981 Zieve, 1981). Similar sequences do not appear to be present in leader RNAs transcribed from 5 -DI particles (Fig. 2), possibly explaining why DI particles do not possess the capacity to inhibit cellular RNA synthesis (Week and Wagner, 1978). 

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