Analysis ofCis-Acting Elements in the 5′ Leader Sequence of Alfalfa Mosaic Virus RNA 3

Abstract

The leader sequence of RNA 3 of the Leiden isolate of alfalfa mosaic virus strain 425 consists of 345 nucleotides (nt) and contains four putative stem–loop structures each with a motif in the loop that resembles the internal control region 2 (ICR2) of tRNA genes. The sequence of the 5′ terminal 112 nt of this leader contains one of these stem–loop structures and is sufficient for a reduced accumulation of RNA 3 in protoplasts and a delayed accumulation in plants (E. A. G. van der Vossenet al., Nucleic Acids Res.21, 1361–1367 (1993). A number of mutations were made in this 112-nt leader sequence to investigate its role in RNA 3 accumulation. Deletion of nucleotides 23–43, 44–90, or 55–112 and inversion or duplication of nucleotides 44–90 all abolished RNA 3 accumulation. Similarly, two base substitutions in the ICR2 motif (nucleotides 60–77) abolished RNA 3 accumulation. Mutations in the stem sections of the putative stem–loop structure had various effects on RNA 3 accumulation and supported the notion that this structure is important for plus-strand promoter activity.