Figure 1.
Identification of promoter activities in E. coli.
(A) A schematic representation of the genomic map of AYVV-NT and the strategy used to randomly amplify fragments of AYVV-NT genome. The positions and directions of the known ORFs are indicated by the grey arrows. Random PCR products were cloned into the promoter trapping vector, pGlow-TOPO® to identify the fragments with potential prokaryotic promoter activities in E. coli. The position of AV3 promoter is indicated by open box. (B) Examination of colonies with a handheld UV illuminator at 365 nm. The fluorescent colonies are indicated by the white arrowheads. (C) One selected colony (as indicated) was grown in the LB medium and examined under UV illumination.
Figure 2.
Fine mapping of the essential AV3 promoter region.
Bar chart representation of the quantification of GFP fluorescence intensities of various constructs. The average fluorescence intensities of four replicates from pGP613-887 were used as the basis for comparison, with those from other constructs expressed as relative fold changes (Y axis). The identities of the constructs are indicated at the bottom of each bar. The error bars indicate standard deviations calculated from at least four replicates.
Figure 3.
Characterization of AV3 promoter activities.
(A) Schematics of the constructs used for the analyses of polarity, putative endogenous RBS and the translatability of downstream ORFs. The identities of the constructs are as indicated on the left, and the GFP reporter gene on the vector is shown as the hashed box. Promoter regions are represented by the open boxes, with the arrows heads indicating the orientation relative to the AYVV-NT virion-sense strand. The grey and black bars in the promoter regions represent the artificially added RBS and the endogenous RBS, respectively. The expected translation start codons and the mutated stop codons on pGP762-889GFP and pGP762-1062GFP are indicated by the triangles and asterisks, respectively. (B) Characterization of the AV3 promoter by fluorescence quantification as described in Figure 2. (C) Nucleotide sequence of the AYVV-NT AV3 promoter region under study, and the respective translation products of two putative downstream ORFs. The relative nucleotide positions are indicated by the numbers on the sides. The putative endogenous RBS (-AGGA-), and the start codons (ATG) for the two downstream ORFs are boxed and underlined, respectively. The two downstream ORFs, which are in different frames, with the first and the second ORF in frame +3 and +2, respectively, relative to nt 1 of the AYVV-NT genome, as denoted by f+3 and f+2. The amino acid sequences of the translation products of each ORF are shown in one-letter code underneath, with an asterisk indicating the stop codon. The region containing the putative -35 and -10 boxes that was used for sequence analyses (Table S2) is italicized. (D) Analysis of GFP reporter mRNA accumulation by northern blot hybridization. Total nucleic acids from E. coli harboring different constructs (as indicated at the top) were separated by electrophoresis through a 1% agarose gel, and stained by EtBr or transferred to a NC membrane followed by hybridization with DIG-labeled probes specific to GFP mRNA. The positions of the ribosomal RNAs (rRNA), the plasmid DNAs, and GFP mRNAs are indicated.
Figure 4.
Translation of the putative downstream ORFs as detected by western blot analysis.
Total proteins from E. coli harboring various constructs (as indicated at the top) were separated by electrophoresis through a 12.5% polyacrylamide gel containing 1% SDS, transferred to a PVDF membrane, and probed with a GFP-specific antiserum. The relative molecular weight of the size marker (lane M) is shown on the left. The position of GFP is indicated on the right. A non-specific protein band is indicated by an asterisk. The expected molecular weight of the GFP-fusion protein expressed from pGP762-889GFP and the positive control from pGP762-869 are 28.96 kDa and 28.62 kDa respectively, as calculated from the amino acid sequence.
Figure 5.
Comparison of promoter strengths.
The strengths of different AV3 promoter regions (as indicated) were compared to that of the constitutive promoter, rrnB P1, and to the promoter for the Rep gene by fluorescence quantification as described in Figure 2.
Figure 6.
Analysis of promoter activities in the regions corresponding to the AYVV-NT AV3 promoter in various begomoviruses.
(A) Comparison of nucleotide sequence similarities among the AV3 promoter corresponding regions in different begomoviruses. The respective nucleotide positions are indicated by numbers on the right. The consensus sequence of prokaryotic promoter containing the -35, -10 regions (in red boxes) are shown above the alignment for comparison. Nucleotide sequences identical to that of the AYVV-NT are represented by a dash. Conserved sequences among all sequences are indicated by an asterisk under the alignment. The putative endogenous RBS (-AGGA-), and the start codon (ATG) of the AYVV-NT sequence are in black box and underlined, respectively. (B) Relative promoter activities of the AV3 promoter corresponding regions from various begomoviruses were quantified as described in Figure 2.