To explore the functionality and conservation of specific base differences in the 3' 200 nucleotides of brome mosaic virus (BMV) RNA-1 (1t) and RNA-2 (2t) with respect to the 3' end of RNA-3 (3t), all possible permutations were used to exchange these regions among the genomic RNAs. When all RNAs bore the 1t promoter, total RNA accumulation was only 15% of wild type; when the 2t or 3t promoter was present on all three RNAs total RNA accumulation was reduced to 30 or 35% of wild type. Two major processes were found to be involved in these dramatic differences. The first reflects the distinct and competitive strengths of the (-)-strand promoters in these sequences, which were shown to have a 3t greater than 1t greater than 2t hierarchy. The second is the importance of the context of upstream sequences in which the 3' promoter is placed. Important contributions of the 3t promoter in preferential amplification of RNA-3 were apparent from changed RNA 1 + 2 3 ratios and reduced progeny accumulation from transfections using the RNA-3/1t chimera. These interactions contribute to temporal modulation as well as overall optimization of viral RNA functions, leading to selection and maintenance of the specific base differences present in the otherwise highly conserved 3' 200 nucleotides of each genomic RNA component of BMV.