Directed mutagenesis as a technique to study protein function: application to β-lactamase

The function of a protein follows uniquely from its three-dimensional structure, which is unambiguously determined by the linear sequence of amino acids. Thus to undertake a systematic study of the relationship between protein structure and function, one would ideally like to be able to alter the structural gene in various ways to encode proteins with novel sequences, structures and functions. Various mutagenic strategies and methods have recently been developed that allow one to achieve these objectives

Proteins to Order Use of Synthetic DNA to Generate Site-Specific Mutations

The ability to cause specific changes in the amino acid sequences of proteins would greatly advance studies on the influence of protein structure on biochemical function. If the desired changes can once be made in the nucleic acid which encodes the protein, one can use cloning in an appropriate microorganism to produce essentially limitless quantities of the mutant protein. We describe here the application of oligonucleotide-directed site-specific mutagenesis to accomplish this objective for the enzyme B-lactamase, the gene for which is contained in the plasmid pBR322. The method uses a procedure to screen for mutant clones which depends on the DNA in the various colonies and not on the properties of the mutant protein; the method can, therefore, be widely applied and does not require, in each separate case, the development of a screening procedure which depends on some phenotypic difference between mutant and wild-type protein

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5′ end binding factors, 3′ end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date

Processing of chloroplast ribosomal RNA transcripts in Euglena gracilis bacillaris

The ribosomal RNA operons ( rrn operons) of Euglena gracilis chloroplasts contain genes for (in order) 16S rRNA, tRNA Ile , tRNA Ala , 23S rRNA and 5S rRNA. Major sites of cleavage of the primary rrn transcript were identified by Northern blot hybridization and S1-mapping. The presumptive termini of all of the mature products have now been identified. During initial processing in the chloroplast, the primary transcript is cleaved between the two tRNAs and between the 23S and 5S rRNAs so as to separate the sequences found in the different mature rRNAs. Subsequently the tRNAs are separated from the rRNAs, further trimming provides the remaining proper ends, and the 3′-ends of the tRNAs are added.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46969/1/294_2004_Article_BF00419917.pd

PRP5: a helicase-like protein required for mRNA splicing in yeast.

A 96-kDa protein predicted by the DNA sequence of the Saccharomyces cerevisiae PRP5 gene contains a domain that bears a striking resemblance to a family of RNA helicases characterized by the conserved amino acid sequence Asp-Glu-Ala-Asp (D-E-A-D). Previous work indicated that the product of the PRP5 gene is required for splicing and that spliceosome assembly does not occur in its absence. However, its precise role in splicing and the nature of its biochemical activity remained unknown. To examine the role of PRP5 in splicing, we cloned the gene by complementation of a temperature-sensitive mutation and determined its DNA sequence. We discuss here the possible roles for an RNA helicase in splicing and for the activity of the PRP5 protein

Studies of Protein Function by Various Mutagenic Strategies: β-Lactamase

Structure unambiguously determines the function of proteins; the linear amino acid sequence in turn dictates three-dimensional structure. Important insights into the molecular origins of the functional behavior of a particular protein become possible if one can alter in prescribed ways the structure of that protein, thereby allowing one to undertake structure-function studies of the type that have a long history of success in organic and pharmaceutical chemistry. Changes in the structure of a protein can be most expeditiously accomplished by appropriate changes in the base sequence of the DNA encoding that protein

Creation of a test plasmid for detecting G-C-to-T-A transversions by changing serine to arginine in the active site of beta-lactamase.

Oligonucleotide-directed mutagenesis of the beta-lactamase gene, bla, on pBR322 was used to change the codon for the active-site serine 70, AGC, to CGC, coding for arginine. Escherichia coli cells carrying the mutant plasmid, pGD104, were sensitive to ampicillin, indicating that the arginine-containing enzyme is inactive. We characterized the reversion of the mutant bla gene by a number of mutagens and in different genetic backgrounds and demonstrated that full ampicillin resistance can be restored only by a G-C-to-T-A transversion occurring at the first base of the codon. Thus, reversion of the mutant bla gene is diagnostic for G-C-to-T-A transversions, and bacteria carrying pGD104 can be used as test strains to detect the occurrence of this mutation

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function.

We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322. Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71). By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype. This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization. Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant