Contribution of downstream promoter elements to transcriptional regulation of the rice tungro bacilliform virus promoter

Abstract

Downstream sequences influence activity of the rice tungro bacilliform virus (RTBV) promoter in protoplasts derived from cultured rice cells. We previously identified a DNA element located between positions +50 and +90 relative to the transcription start site to which rice nuclear proteins bind. In this study, using DNA UV crosslinking assays, we show that two rice nuclear proteins bind specifically to this DNA element. We demonstrate that the DNA element enhances RTBV promoter activity in a copy number-dependent manner when transferred to a position upstream of the promoter. In addition, using electrophoretic mobility shift assays, we show that at least two novel nuclear proteins from rice cell suspension cultures bind to a subregion (from +50 to +59) of the DNA element and that a protein from rice root, but not shoot, nuclear extracts interacts with a perfect palindromic sequence motif located within the sequence +45 to +59. Furthermore, a position-dependent GAGA motif, present in three copies within downstream promoter sequences from +1 to +50, is involved in the regulation of RTBV promoter activity.

Figure 1

Analyses of the interactions between dps from +1 to +90 and proteins in different types of rice nuclear extracts. EMSAs were performed using a labeled DNA probe from +1 to +90. (A) DNA fragments (dps, P1 or P2) and double-stranded oligonucleotides having overlapping sequences between +1 and +90 (dps1 and dps-a to dps-c) were used as cold competitors at a 200-fold molar excess in EMSAs. (B) EMSAs were performed without (lane 1) or with nuclear extracts from cell suspensions or shoots in the absence (–) or presence of competitors (dps, dps1, P1 or P2). DNA–protein complexes (C1 and C2) are indicated. P is free DNA probe. (C) EMSAs were carried out without (lanes 1 and 6) or with cell suspension or root nuclear extracts in the absence (–) or presence of competitors (dps, dps1 or dps-a to dps-c). DNA–protein complexes are designated C1–C3.

Figure 2

Mutational analysis of the minimal binding site of complex C1 on dps1. (A) Wild-type dps1 and mutants were used as competitors at a 200-fold molar excess in EMSAs. Dashes indicate identity with the wild-type sequence. Only the mutated bases are indicated. (B) EMSA experiment with labeled DNA probe from +1 to +90 in the absence (lane 1) or presence (lanes 2–12) of nuclear extract from rice shoots. Competitors used are indicated at the top of the gel.

Figure 3

Copper-phenanthroline footprinting analysis. (A) A DNA fragment covering +1 to +150 was 5′-end-labeled on either the top or bottom strand and incubated with rice shoot (S) or cell suspension (C) nuclear extracts. The mixture was resolved on a native polyacrylamide gel. DNA–protein complex C1 and free probe (F) were then digested in situ with 1,10-phenanthroline-copper ion. DNAs were eluted from the gel and resolved on a 6% polyacrylamide sequencing gel. A Maxam–Gilbert sequencing ladder (G) was run in parallel. The protected areas are depicted on the right of the gel by vertical bars. Numbers correspond to base pairs downstream of the transcription start site. The hypersensitive sites are indicated with arrowheads. (B) Nucleotide sequence of dps1 showing the areas protected in complex C1 (open boxes).

Figure 4

Mutational analysis of the minimal binding sites of complexes C2 and C3 on dps2. (A) Wild-type dps2 and mutants used as competitors at a 200-fold molar excess in EMSAs. Dashes in the sequences indicate identity with the wild-type sequence. Only the mutated bases are indicated. (B) EMSAs were performed with labeled DNA probe from +1 to +90 in the absence (lane 1) or presence (lanes 2–17) of cell suspension nuclear extracts. Competitors used are indicated at the top of the gels. DNA–protein complexes are indicated. (C) EMSAs were performed as in (B) but with root nuclear extracts. (D) Nucleotide sequence of dps2. Open boxes with solid and dotted lines represent the binding sites of complexes C2a and C2b, respectively. Gray boxes indicate binding sites of complex C3. Arrows depict palindromic sequences.

Figure 5

Analysis of the DNA-binding proteins by DNA UV crosslinking. A radiolabeled DNA probe substituted with BrdU was incubated without (lane 1) or with nuclear extracts from rice shoots (S) or cell suspensions (C) in the absence (–) or presence of a 100-fold molar excess of the competitors indicated. UV crosslinked proteins were separated in a 14% SDS–polyacrylamide gel. The sizes of the marker proteins are indicated on the right. The arrowheads indicate the apparent molecular masses of the crosslinked proteins. Asterisks indicate incomplete digestion of the labeled DNA.

Figure 6

dps1 enhances RTBV promoter activity from an upstream position. Constructs used for transfection of O.sativa protoplasts are shown on the left. The filled box indicates dps1 (+50 to +90). Dotted boxes depict deletions of dps1. The dotted line represents deletion of dps3 (+8 to +50). A bent arrow indicates the transcription start site. All constructs were tested in at least three independent transfections. For each construct the mean promoter activity is indicated as a percentage of the activity of the wild-type construct R-218 (set as 100%).

Figure 7

Deletion analysis of the RTBV leader sequences. Constructs consisting of the RTBV upstream promoter sequence (to position –218) and the RTBV leader sequence (either complete or truncated as indicated) are shown schematically. All constructs were tested in transfected O.sativa protoplasts in at least three independent experiments. For each construct the mean promoter activity is indicated as a percentage of the activity of the wild-type construct R-218 (set as 100%).

Figure 8

GAGAG motifs contribute to RTBV promoter activity. (A) Nucleotide sequence and potential secondary structure of the dps3 transcript. (B) Schematic representation of the constructs tested in transfected O.sativa protoplasts showing the expanded dps3 region with deletions. A filled box indicates the TATA box. A bent arrow represents the transcription start site. Open boxes show the GAGAG motifs. Dashes in the sequences indicate identity with the wild-type sequence. Asterisks indicate deleted bases. Constructs and relative CAT activities are shown on the left. The relative CAT activity given for each construct is an average of at least three independent transient expression assays.

Figure 9

Effect of the spacing between the initiation site and the GAGAG motif on promoter activity. A schematic representation of the constructs tested in transfected O.sativa protoplasts is shown at the top, with the expanded sequence underneath indicating insertions between +10 and +11. Inserted nucleotides are shown in parentheses. Corresponding CAT activities are shown on the left. The results shown are the average of at least three independent transient expression assays.