Ribosomes with large synthetic N-terminal extensions of protein S15 are active in vivo.
The genes for ribosomal proteins S4, S13 or S15 were fused with the gene for staphylococcal protein A, or derivatives thereof (2A'-7A'). The gene fusions were introduced into Escherichia coli strains, mutated in the corresponding ribosomal protein gene, by transformation. These mutated ribosomal proteins cause a phenotype that can be complemented. Thus, the phenotype of the transformants was tested and the ribosomal proteins were analyzed. The S4 N-terminal fusion protein severely disturbed growth of both the mutant and the wild-type strains. The S13 C-terminal fusion protein was proteolyzed close to the fusion point, giving a ribosomal protein moiety that could assemble into the ribosome normally. S15 N-terminal fusion proteins complemented a cold-sensitive strain lacking protein S15 in its ribosomes. These fused proteins were assembled into active ribosomes. The position of S15 in the 30S ribosomal subunit is well known. Therefore, in structural studies of the ribosome in vivo, the S15 fusion proteins can be used as a physical reporter for S15.
Walles-Granberg
A
A
Department of Microbiology, Stockholm Univeristy, Sweden.
Schnell
R
R
Isaksson
L A
LA
Rydén-Aulin
M
M
D001483:Base Sequence; D002853:Chromatography, Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D013205:Staphylococcal Protein A; C036004:ribosomal protein S15