Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1.

The expression of the Arabidopsis thaliana PAT1 gene, which encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase, was investigated using translational fusions of the PAT1 promoter to the GUS reporter gene. Independent stably transformed A. thaliana lines containing a single copy of a fusion that includes the entire plastid transit peptide and the first two introns of PAT1 had on average 30 times more GUS enzyme activity than plants transformed with a construct in which GUS was fused a short distance downstream of the PAT1 start codon. Plants containing the construct without introns or leader peptide accumulated undetectable amounts of PAT1-GUS fusion protein and mRNA, even though the transcriptional rate of both fusion constructs was comparable. Fusions containing the entire transit peptide and either of the first two introns yield as much GUS activity as constructs containing both introns, but constructs containing the transit peptide but no introns give rise to much lower levels. Therefore, introns greatly enhance the expression of PAT1-GUS fusions, and they act post-transcriptionally to increase the steady-state level of mRNA.

Immunological characterization and chloroplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thaliana.

In order to study the tryptophan biosynthetic enzymes of the plant Arabidopsis thaliana, polyclonal antibodies were raised against five of the tryptophan biosynthetic pathway proteins: anthranilate synthase alpha subunit, phosphoribosylanthranilate transferase, phosphoribosylanthranilate isomerase, and the tryptophan synthase alpha and beta subunits. Immunoblot analysis of Arabidopsis leaf protein extracts revealed that the antibodies identify the corresponding proteins that are enriched in Arabidopsis chloroplast fractions. Precursors of phosphoribosylanthranilate isomerase and tryptophan synthase alpha subunit were synthesized by in vitro translation. The precursors were efficiently imported and processed by isolated spinach chloroplasts, and the cleavage sites within the precursors were determined. These results provide the first direct evidence that the tryptophan biosynthetic enzymes from Arabidopsis are synthesized as higher molecular weight precursors and then imported into chloroplasts and processed into their mature forms.

The importance of surface loops for stabilizing an eightfold beta alpha barrel protein.

An important step in understanding how a protein folds is to determine those regions of the sequence that are critical to both its stability and its folding pathway. We chose phosphoribosyl anthranilate isomerase from Escherichia coli, which is a monomeric representative of the (beta alpha)8 barrel family of proteins, to construct a variant that carries an internal tandem duplication of the fifth beta alpha module. This (beta alpha)9 variant was enzymically active and therefore must have a wild-type (beta alpha)8 core. It had a choice a priori to fold to three different folding frames, which are distinguished by carrying the duplicated segment as an insert into one out of three different loops. Steady-state kinetic constants, the fluorescence properties of a crucial tryptophan residue, and limited proteolysis showed that the stable (beta alpha)9 variant carries the insertion between beta-strand 5 and alpha-helix 5. This preference can be explained by the important role of loops between alpha helices and beta strands in stabilizing the structure of the enzyme.

Isolation and characterization of mutations creating high-efficiency transcription initiation signals within the trp operon of Escherichia coli.

Two different mutational events generate promoter-active deoxyribonucleic acid sequences within the trp operon of Escherichia coli, probably through single base-pair changes. The mutations, obtained after ethyl methane sulfonate mutagenesis by selecting for elevated lac gene expression in a trp-lac fusion, are cis dominant and trans recessive with respect to their effects on the synthesis of downstream enzymes. One of the mutants (trpD11), obtained repeatedly under the selective conditions employed, prevents the formation of active phosphoribosyltransferase. The second mutation, trp3B, has no effects on any trp enzyme. By deletion mapping, trpD11 was localized near the operator-distal end of trpD, outside the segment of deoxyribonucleic acid that contains the low-efficiency internal promoter trpP2. Reversion to prototrophy of trpD11 was greatly stimulated by 2-aminopurine and ethyl methane sulfonate. Tests with suppressors indicated that trpD11 is a UAA (ochre) nonsense mutation. Under repression conditions, strains harboring either lesion in a normal trp operon synthesize the tryptophan biosynthetic enzymes in a noncoordinate fashion. The products of the operator-distal structural genes trpC, trpB, and trpA are formed at rates approximately 15-fold higher than those of wild type. The enzymes encoded by operator-proximal genes trpE and trpD are low or not detectable. Under derepression conditions, coordinate expression of the operon was observed.

Regulation of enzyme synthesis in the tryptophan pathway of Acinetobacter calcoaceticus.

In Acinetobacter calcoaceticus the seven genes coding for the enzymes responsible for tryptophan synthesis map at three chromosomal locations. Two three-gene clusters, one (trpGDC) specifying the small subunit of anthranilate synthase, phosphoribosyl transferase, and indoleglycerol phosphate synthase and the other (trpFBA) specifying phosphoribosyl anthranilate isomerase and both tryptophan synthase subunits, are not linked to each other or to the trpE gene specifying the large anthranilate synthase subunit. When regulation of trp gene expression is studied in the wild type, only the level of the trpF gene product decreases upon addition of tryptophan to the medium. Tryptophan starvation of tryptophan auxotrophs, however, results in increased levels of all the tryptophan enzymes; this and additional evidence suggests that the expression of all the trp genes is subject to repression. The trpGDC genes are coordinately controlled, and the trpE gene is regulated in parallel with them. The trpFBA genes are controlled neither coordinately nor in parallel with the other trp genes, but respond proportionally when compared with each other. So far, two types of constitutive mutants have been found. The first class of mutants apparently occurs in the structural gene for a repressor protein; this repressor locus is unlinked to any of the biosynthetic trp genes and affects only the expression of trpE and the trpGDC cluster. The second class contains mutants closely linked to the trpGDC region; they overproduce only the gene products of this cluster.