The amino terminal region delimited by Met1 and Met37 is an integral part of the 32 kDa herbicide binding protein

Abstract

The 33.5 kDa precursor of the 32 kDa herbicide binding protein is encoded in the chloroplast by the psbA gene. Previous work on the gene and protein did not pinpoint the initiation site of translation in vivo. To resolve this issue, constructs containing full length, truncated or mutated psbA from Solanum nigrum were used as templates for in vitro transcription in an SP6-promoter directed system. The resulting RNAs were then translated in a reticulocyte lysate system. The SDS-PAGE and partial proteolysis patterns of the translation products demonstrate that constructs containing the first ATG codon (Met1) of the psbA open reading frame code for a full length precursor protein (33.5 kDa), irrespective of whether the second ATG codon (Met37) is replaced by site directed mutagenesis or not. However, a 5' deleted gene, lacking the codon for Met1 but containing the codon for Met37, codes for a truncated 29 kDa protein. It is concluded that the 33.5 kDa precursor protein and its 32 kDa maturation product initiate at Met1 of the psbA open reading frame.

Expression of the barley psbA gene in Escherichia coli yields a functional in vitro photosystem II protein D1

“…Moreover, the psbA gene contains a sequence similar to a prokaryotic Shine-Dalgarno sequence just upstream of the Met-37 codon. Our results correlate with data obtained earlier on the expression of psbA genes of other higher plants in prokariotic in vitro translation systems [3,14]. At the same time, in our experiments on in vitro expression of the barley psbA gene in the rabbit reticulocyte lysate system, a major expression product of A& -33.5 kDa, and only a small amount of 29 kDa protein was observed [6].…”

“…In higher plants, Dl protein (or herbicide-binding protein) is coded by the psbA gene located in the chloroplast genome as a single copy [2]. In chloroplasts this protein first appears as a precursor form, which has an apparent M, of 33.5 kDa, and is then processed to a mature form of M, 32 kDa [3].…”

Characterization of a four-member psbA gene family from the cyanobacterium Anabaena PCC 7120

“…When a primer extension assay was performed with an oligonucleotide specific for psbAII, III and IV transcripts, one major and several minor products were observed suggesting that one or more of the psbAII-IV genes is transcribed. The major product and two of the minor products differ by a few bases in length and map downstream from the region where the E coli - 10 At a similar distance downstream from all four psbA genes are long inverted repeats potentially capable of forming stable stem and loop structures in the transcription product (Fig. 5).…”

In vitro transcription and translation of the psbD gene encoding the D-2 protein of photosystem II in barley

“…c) The 5' leader sequence of the psbD gene in barley, spinach, tobacco, liverwort and pea contains a conserved TAA and TAG stop codon in position -21 from the initiation codon (21), that may prevent translation in the E. coli lysate. Such a stop codon is also observed in the 5' leader sequence of the psbA gene of pea (23) and tobacco (33) at positions -18 and -21, respectively, and may be responsible for the fact that translation of in vitro-generated psbA mRNA in the E. coli lysate is initiated only at the second methionine codon (4) and not at the first as is the case in the chloroplast (6).…”