Characterization of the Maize Mutator Transposable Element MURA Transposase as a DNA-Binding Protein

Abstract

The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved ϳ32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu The Mutator family of transposable elements of Zea mays is considered to be one of the most efficient gene-tagging systems in eukaryotes. Mutator (Mu) transposable elements were first recognized by Robertson in a line of maize that exhibited a 50-to 100-fold increase in its spontaneous mutation frequency compared to standard maize stocks (45). Extensive genetic and molecular analysis has demonstrated that the increased mutation frequency is caused by the transposition of a family of Mu elements (reviewed in references 10, 13, and 16). Molecular analysis of Mu-induced mutations has identified at least eight classes of nonautonomous and autonomous Mu elements (10). These elements share highly conserved ϳ210-bp terminal inverted repeats (TIRs), and their insertion sites are flanked by a 9-bp direct repeat duplication of the host sequence. Elements are classified by the sequence similarity of their internal sequences. Mu1 is the most active Mu element as measured by frequency of insertion into tagged and cloned maize genes (13). Mu1 elements are also among the smallest of the Mu elements; they have ϳ210-bp TIRs encompassing an ϳ1,000-bp internal region (5). Mu1, like most of the elements in the Mu family, is a nonautonomous element that depends on the presence of MuDR (also called Mu9), the autonomous element, for its transposition (20,28,29,43).MuDR is a 4.9-kb element with characteristic Mu TIRs of ϳ210 bp, but it is unique in encoding proteins required for transposition. MuDR encodes two major transcripts, mudrA and mudrB, that are convergently transcribed from promoters in each of the TIRs. The mudrA and mudrB transcripts can be differentially spliced (27). It is not yet known if these splicing variants produce all of the predicted alternate protein products and, if so, what roles these protein products play in Mutator activities. This study focuses on the 823-amino-acid open reading frame, MURA, encoded by the major, fully spliced mudrA transcript (27). MURA is of particular interest because it shares a sequence motif with a family of bacterial insertion sequence transposases (22)...

Jittery, a Mutator Distant Relative with a Paradoxical Mobile Behavior: Excision without Reinsertion

“…Interestingly, given the total lack of MURB-related sequences in the databases, no sequences similar to TnsE are found among Tn7 relatives in other bacteria. In the absence of JITB, JITA would still bind to sites in the Jit TIRs, analogous to the MURA binding sites found in the MuDR TIRs (Benito and Walbot, 1997), and make staggered cuts in the DNA immediately adjacent to the transposon ends. However, the resulting double-strand break would be subsequently repaired without footprint formation by the action of an endogenous ligase present in all maize lines, and, in the absence of a cleaved receptor site, Jit would fail to reinsert in the genome.…”

Sequence diversity and copy number variation of Mutator-like transposases in wheat

“…Furthermore, the results also suggest that the transposition of each MULE subclass is under a different regulation. The MURA transposase binding site (MBS), a 32-bp motif in the TIRs, is well conserved among the mobile Mutator elements (Benito and Walbot 1997;Rudenko and Walbot 2001). It was suggested that transposase active for transposition of subclass II MoTUs might not be able to recognize MBSs of subclass I MoTUs.…”

“…MuDR carries two genes, mudrA and mudrB. The former encodes the MURA transposase that catalyzes the excision of Mutator (Eisen et al 1994;Benito and Walbot 1997) and the latter encodes the MURB protein that is proposed to be involved in the reinsertion of Mutator (Lisch et al 1999;Raizada and Walbot 2000). However, the sequence corresponding to mudrB has only been identified in the genus Zea so far (Lisch et al 1995;Walbot and Rudenko 2002).…”

Epigenetic silencing and unstable inheritance of MuDR activity monitored at four bz2-mu alleles in maize (Zea mays L.)

“…MuDR contains two protein-coding genes: mudrA encodes MURA transposase and mudrB encodes MURB helper proteins functioning in the Mu insertional activity (Lisch et al, 1999;Raizada and Walbot, 2000). All Mu elements share ~215-bp terminal inverted repeat (TIR) sequences, and the mobile Mu elements contain a highly conserved 32-bp MURA transposase binding site within both TIRs (Benito and Walbot, 1997). Only a few maize lines contain active MuDR elements, but all maize lines examined carry homologous MuDR sequences (hMuDR elements; Rudenko and Walbot, 2001).…”