Identification and characterization of transcription factor IIIA and ribosomal protein L5 from Arabidopsis thaliana

Abstract

Thus far, no transcription factor IIIA (TFIIIA) from higher plants has been cloned and characterized. We have cloned and characterized TFIIIA and ribosomal protein L5 from Arabidopsis thaliana. Primary sequence comparison revealed a high divergence of AtTFIIIA and a relatively high conservation of AtL5 when compared with other organisms. The AtTFIIIA cDNA encodes a protein with nine Cys(2)-His(2)-type zinc fingers, a 23 amino acid spacer between fingers 1 and 2, a 66 amino acid spacer between fingers 4 and 5, and a 50 amino acid non-finger C-terminal tail. Aside from the amino acids required for proper zinc finger folding, AtTFIIIA is highly divergent from other known TFIIIAs. AtTFIIIA can bind 5S rDNA, as well as 5S rRNA, and efficiently stimulates the transcription of an Arabidopsis 5S rRNA gene in vitro. AtL5 identity was confirmed by demonstrating that this protein binds to 5S rRNA but not to 5S rDNA. Protoplast transient expression assays with green fluorescent protein fusion proteins revealed that AtTFIIIA is absent from the cytoplasm and concentrated at several nuclear foci including the nucleolus. AtL5 protein accumulates in the nucleus, especially in the nucleolus, and is also present in the cytoplasm.

Figure 1

Primary sequence alignment of known TFIIIAs with Arabidopsis TFIIIA. Sequences were aligned manually to match each of the nine zinc fingers of AtTFIIIA with the corresponding finger of the other TFIIIAs. Zinc fingers are framed and numbered. Note that the ninth finger from S.cerevisiae was aligned with finger 10 from S.pombe only for illustration. Non-aligned regions between fingers 1 and 2, and 4 and 5 in the AtTFIIIA sequence are underlined. AtTFIIIA zinc fingers are in red, and zinc fingers of other organisms are shown in blue. Conserved residues (in >50% of the sequences) in the zinc fingers or in non-finger regions are highlighted in yellow or gray, respectively. The oocyte motif MGEK/R and the NES motifs are highlighted in green and blue, respectively. NLS motifs are written in green characters, and TAS motifs are indicated in black bold type. Asterisk marks the end of the sequence. The residue number is shown to the right of each line of sequence.

Figure 2

Primary sequence alignment of AtL5 with some known L5s. Sequences were aligned using the CLUSTALW program. Identical residues (in 100% of the sequences) are highlighted in black, and conserved residues (in >50% of the sequences) are highlighted in gray. The residue number is shown to the right of each line of sequence.

Figure 3

DNA binding assays of GST–AtTFIIIA and GST–AtL5 recombinant proteins. (A and D) SDS–PAGE of GST–AtTFIIIA and GST–AtL5 recombinant proteins, respectively. Shown are results for uninduced E.coli BLR (DE3) (lane 1), IPTG-induced cells (lane 2) and purified recombinant proteins (lane 3). Proteins were visualized by staining with Coomassie brilliant blue. The sizes (in kDa) of molecular mass markers run in lane M are indicated on the left. Increasing concentrations (1–170 nM) of unlabeled 5S rDNA (B) or non-specific competitor DNA (C) were added to binding reactions including labeled 5S rDNA and GST–AtTFIIIA (2.5 ng/µl). Binding reactions were performed with increasing concentrations (2.5– 10 ng/µl) of either AtL5 (E) or GST alone (F). Arrowheads indicate free (unbound) probe, and protein–DNA complexes are indicated by an asterisk. n.p., no protein.

Figure 4

DNase I footprint of AtTFIIIA. DNase I cleavage of each strand is presented. Numbers to the right of each gel indicate the position of DNase I cleavage relative to the start site of transcription (+1). The positions of the A-box, the intermediate element (IE) and the C-box are shown to the left of each gel.

Figure 5

5S rRNA binding analysis of GST–AtTFIIIA and GST–AtL5 proteins. A constant amount of labeled 5S rRNA probe synthesized in vitro was incubated with increasing concentrations (2.5–10 ng/µl) of GST–AtL5 (A), GST–AtTFIIIA (B) or GST (D) proteins and then subjected to gel mobility shift analysis. As a control, GST–AtTFIIIA and GST–AtL5 proteins were incubated with non-specific RNA (C). Arrowheads indicate free (unbound) probe, and protein–RNA complexes are indicated by an asterisk. n.p., no protein.

Figure 6

In vitro transcription assays. (A) Reactions were performed without added recombinant protein (n.p.) or with increasing concentrations (2.5–7.5 ng/µl) of GST–AtTFIIIA or GST. (B) Quantification and graphical representation of the data from several independent experiments similar to that shown in (A). The asterisk and arrowhead indicate endogenous tRNA and 5S rRNA, respectively. Molecular sizes are indicated (in nucleotides) next to the gel.

Figure 7

Cellular localization of AtTFIIIA and AtL5 proteins. Protoplasts were transformed with constructs expressing the proteins indicated and were observed 30 h post-transfection (B, D and F). Corresponding differential interference contrast (DIC) images are shown in (A), (C) and (E), respectively. White arrowheads indicate the nucleolus. (G and H) Close-up views of two protoplasts nuclei expressing the GFP–AtTFIIIA fusion protein. Small arrowheads indicate some of the bright nuclear foci present in addition to the nucleolus (large arrowhead). Scale bar (A–H) 10 µm.