Abstract
Functional primordial proteins presumably originated from random sequences, but it is not known how frequently functional, or even folded, proteins occur in collections of random sequences. Here we have used in vitro selection of messenger RNA displayed proteins, in which each protein is covalently linked through its carboxy terminus to the 3' end of its encoding mRNA, to sample a large number of distinct random sequences. Starting from a library of 6 x 1012 proteins each containing 80 contiguous random amino acids, we selected functional proteins by enriching for those that bind to ATP. This selection yielded four new ATP-binding proteins that appear to be unrelated to each other or to anything found in the current databases of biological proteins. The frequency of occurrence of functional proteins in random-sequence libraries appears to be similar to that observed for equivalent RNA libraries.
MeSH Terms
ATP-Binding Cassette Transporters
Adenosine Triphosphate/metabolism
Amino Acid Sequence
Binding Sites
Carrier Proteins/genetics,metabolism
Escherichia coli
Escherichia coli Proteins
Gene Library
Maltose-Binding Proteins
Molecular Sequence Data
Monosaccharide Transport Proteins
Peptide Library
Protein Binding
Protein Folding
Proteins/genetics,isolation & purification,metabolism
RNA/genetics
Recombinant Fusion Proteins/genetics,metabolism
Chemicals
ATP-Binding Cassette Transporters
Carrier Proteins
Escherichia coli Proteins
Maltose-Binding Proteins
Monosaccharide Transport Proteins
Peptide Library
Proteins
Recombinant Fusion Proteins
maltose transport system, E coli
RNA
Adenosine Triphosphate
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Keefe A D
Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA.
Szostak J W
References (11)
11 references, click to expand
-
Randomization of genes by PCR mutagenesis.
PCR Methods Appl. 1992 Aug;2(1):28-33
PMID: 1490172
-
The function and structure of the metal coordination sites within the glucocorticoid receptor DNA binding domain.
Nature. 1988 Aug 11;334(6182):543-6
PMID: 3043231
-
In vitro selection of functional nucleic acids.
Annu Rev Biochem. 1999;68:611-47
PMID: 10872462
-
RNA-peptide fusions for the in vitro selection of peptides and proteins.
Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12297-302
PMID: 9356443
-
Escherichia coli transcription termination factor rho. II. Binding of oligonucleotide cofactors.
J Biol Chem. 1993 Jul 5;268(19):13947-55
PMID: 8314761
-
Constructing high complexity synthetic libraries of long ORFs using in vitro selection.
J Mol Biol. 2000 Mar 24;297(2):309-19
PMID: 10715203
-
An RNA motif that binds ATP.
Nature. 1993 Aug 5;364(6437):550-3
PMID: 7687750
-
Optimized synthesis of RNA-protein fusions for in vitro protein selection.
Methods Enzymol. 2000;318:268-93
PMID: 10889994
-
Mutational analysis of potential zinc-binding residues in the active site of the enterococcal D-Ala-D-Ala dipeptidase VanX.
Biochemistry. 1997 Aug 26;36(34):10498-505
PMID: 9265630
-
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
PMID: 9254694
-
High-resolution molecular discrimination by RNA.
Science. 1994 Mar 11;263(5152):1425-9
PMID: 7510417