Structural Requirements for Protein Function Studied by in Vitro Mutagenesis on Beta-Lactamase

The study of naturally occurring variants of proteins has been successfully used for a long time to assign roles to structural elements in a protein and to correlate functional requirements with the nature of these structural elements. Advances in techniques for DNA synthesis and DNA sequencing, along with the development of recombinant DNA techniques now allow one to clone the gene for a protein and then to modify it at will. In this way, presumably any and all aminoacid substitutions can be engineered into any protein whose gene has been cloned, characterized and expressed. Beta-lactamase has been used as a model system in which to study the feasibility of the approach, and it has been demonstrated that not only is it possible to introduce specific predetermined changes in the structure of a protein, but that these mutants can in turn serve as substrates for further modifications. An inactive enzyme can be used to search for a broad range of structural requirements by imposing selective conditions that require a function for the survival of the host organism for the mutant protein. Four variants of beta lactamase, one of which is catalytically active, have been obtained by site specific mutagenesis. In the inactive mutants the conserved active site sequence -ser70-thr71- was altred to either -thr70-ser71-, -thr70-thr-71- or -arg-70-thr71-; a variant in which a disulfide bond was removed by mutating one of the only two cysteines in E. coli beta-lactamse to serine was found to be active. In addition, a revertant to activity has been obtained from one of the the inactive mutants (-thr70-ser71-). The revertant is different in aminoacid sequence (-ser70-ser71-) and in some of its properties from the wild type enzyme while still having catalytic activity. No revertants with aminoacid substitutions at a secondary site were found. Both the catalytically active revertant and the mutant lacking the disulfide bridge were found to have reduced thermal stability. The rate of secretion of three of these mutants (ser70→thr, thr71→ser and ser70→thr/thr71→ser) was compared to that of the wild type beta lactamase and no significant differences were found. The -arg70-ser71- mutant will be used to identify chemical mutagens and carcinogens that induce GC to TA transversions by collaborators P. L. Foster and D. Botstein.</p

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

Dead-box proteins: a family affair—active and passive players in RNP-remodeling

DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family

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

Chapter 24. In Vitro Mutagenesis:Relationships in Proteins Powerful New Techniques for Studying Structure-Function Relationships in Proteins

To be able to generate, at will, any sequence of amino acids allows rational study of the relation between protein structure and function. For structure–function studies of proteins, a second powerfully complementary approach that takes advantage of the ability of biological systems to produce a very large number of random structural variants is also discussed in the chapter; these can then be screened for those variants that have a particular function. In this case, a particular function is specified, and then it is determined that the structures, of many millions that can be easily tested, have that function. The procedures useful in this approach are those of random mutagenesis, perhaps directed toward a particular region of the protein. This chapter describes particularly those aspects of mutagenesis that have a direct bearing on the study of protein function. These techniques have also been used to address a large variety of genetic problems as well as to probe structure–function relationships; the more general subject has been discussed extensively. The chapter discusses many techniques that have recently been developed that allow a variety of quite novel approaches to the studies of the relationship between the linear sequence and the function of proteins. Much of the discussion has focused on the procedures for manipulating the DNA to achieve proteins with altered function