Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs

Abstract

Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine-Dalgarno (SD) and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5'-untranslated region (5'-UTR) and DB region. Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5'-UTR and DB region promotes accumulation of NPTII up to approximately 16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII yield ( approximately 23% of TSP) was obtained from a construct in which the T7g10 5'-UTR was linked with the NPTII coding region via a NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.

Figure 1

The E.coli mRNA DB and plastid mRNA DS regions. (A) The 15 nt ADB regions in the E.coli (Ec) 16S rRNA (nt 1469–1483) (2,45) and the cognate DS region in the tobacco plastid (pt) 16S rRNA (nt 1416 and 1430) (46). (B) Complementarity of the mRNA DB and DS regions with the plastid 16S rRNA penultimate stem ADS region. Watson–Crick (lines) and G-U (filled circles) pairs are marked. Nucleotides corresponding to the NheI site and neo coding region are in lower case. The numbers of potential pairs formed with the 15 nt DS region are in parentheses.

Figure 2

Plastid vectors to compare translation efficiency from neo mRNAs with the E.coli DB and plastid DS regions. (A) DNA sequence of Prrn promoters with T7g10 leader derivatives. The Prrn promoter region is underlined; the transcription initiation site is marked by a horizontal arrow; the translation initiation codon (ATG) is in bold; the SD sequence is underlined with a wavy line. The T7g10 DB (Ec) sequence is shown on top; the sequence with the plastid DS region (pt) is duplicated in the middle; the sequence with the NheI site is at the bottom. Plastid and T7g10 sequences are in capital letters; nucleotides added or modified during construction are in lowercase; gaps are marked with dashes. (B) The plastid targeting region of the transformation vectors. The spectinomycin resistance (aadA) and neo genes and plastid genes rrn16, trnV and rps12/7 (46) are shown. The positions of the neo promoter (Prrn) and 3′-UTR (TrbcL) are also marked. The wavy line represents 1.0 kb neo mRNA. Abbreviations for restriction sites: E, EcoRI; S, SacI; N, NheI; X, XbaI; H, HindIII; B, BglII. Restriction sites removed during plasmid construction are in parentheses. (C) Listing of plasmids and the schematic map of their promoter and N-terminal coding regions. For an explanation see (B).

Figure 3

Codon frequency in the neo coding region N-terminus. Values are shown for the Prrn derivatives T7g10+DB/Ec (plasmid pHK38), T7g10+DS/pt (plasmid pHK39) and T7g10 (plasmid pHK40). Fraction is the relative usage frequency of a specific codon compared to the usage frequency of all codons encoding the same amino acid. Triplet/1000 is the frequency of occurrence of a certain triplet per 1000 triplets.

Figure 4

NPTII accumulation in tobacco leaves. (A) Immunoblot analysis to detect NPTII. The amount of TSP (µg) loaded on the SDS–PAGE gel is indicated above the lane. A NPTII dilution series was electrophoresed on the same gel. Lanes are marked with the plasmid name used for plant transformation. A protein sample from wild-type tobacco was also loaded (Wt). (B) Protein gel (20 µg/lane) stained with Coomassie Brilliant Blue R250. Control, NPTII (700 ng). The positions of NPTII and the Rubisco large (LSU) and small (SSU) subunits are marked.

Figure 5

Steady-state levels of neo mRNA in the transplastomic leaves. An aliquot of 4 µg total cellular RNA was loaded per lane. The blots were probed for neo (top) and cytoplasmic 25S rRNA (bottom, loading control). The lanes are designated as in Figure 3.