Mutant analysis of Prevotella sp. plaA-lacZ fusion protein expression in Escherichia coli: support for an essential role of the stem-loop.
This study investigated the involvement of RNA folding in the synthesis of a fusion protein with beta-galactosidase activity. The coding gap region of the Prevotella loescheii adhesin gene plaA was fused in-frame with the Escherichia coli lacZ gene on plasmid pSK105. N-Terminal sequencing of the expressed plaA-lacZ protein indicated that it resulted from translational initiation at a fortuitous ribosomal-binding site within the plaA sequence at nt 570. Specific mutations were introduced in the stem-loop region that precedes the gap sequence. Analysis of stem-loop mutants, together with the introduction of compensatory mutations that restored activity, supports a requirement for stem-loop formation within the plaA sequence preceding the translational initiation site. A mutation reducing the predicted size of the loop, but preserving the stem structure, inactivated fusion protein synthesis. A suppressor mutation predicted to restore the size of the loop restored efficient fusion protein synthesis. In addition, the sequence preceding the translational start site of the plaA-lacZ fusion has several similarities to sequences that function as translational enhancers in prokaryotes. These include a stem-loop structure, an A-U rich region preceding the initiation codon, and a region of homology to 16S rRNA.
Manch-Citron
J N
JN
Department of Oral Biology, University of Missouri-Kansas City, School of Dentistry 64108-2716, USA. jcitron@umkc.edu
Dey
A
A
Ewell
J B
JB
Nguyen
N Y
NY
D018829:Adhesins, Bacterial / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism; D005697:Galactosides; D005798:Genes, Bacterial; D007763:Lac Operon / Q000235:genetics; D037102:Lectins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D018720:Prevotella / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D012329:RNA, Bacterial / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*
D018829:Adhesins, Bacterial; D005697:Galactosides; D037102:Lectins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C007816:beta-galactoside; C086517:plaA protein, Prevotella loescheii