Regulation of both gene expression and protein stability provides genetically assisted target evaluation (GATE) for microbial target validation.

The attempt to develop novel antibiotics, active against organisms resistant to current therapies, has led researchers to seek and explore new drug targets. The rapid sequencing and analysis of entire microbial genomes has identified large numbers of genes that may be sufficiently different from their human counterparts to be exploited as targets for antimicrobial treatment. As a first step, the importance of the various putative targets for microbial growth and survival must be assessed. Emerging validation technologies are becoming increasingly sophisticated and, in certain cases, allow prioritisation of the best targets. In this paper, genetically assisted target evaluation (GATE) is introduced as a versatile target validation technology. GATE concomitantly manipulates both synthesis and stability of the targeted protein using copper ions as an effector. This technology allows rapid quantitation of the lethal consequences of inactivation of targeted gene products in Saccharomyces cerevisiae. Additional tools can then be applied to extend these results into pathogenic organisms, such as Candida albicans.

Preparation of protein extracts from yeast.

Three protocols are presented for preparing protein extracts; they differ primarily in the way the cells are broken. In the basic protocol, cells are enzymatically converted to spheroplasts, which are then lysed by a combination of osmotic shock and Dounce homogenization. A support protocol for isolating intact nuclei by differential centrifugation is also presented. An alternate protocol describes mechanical breakage of cells by vortexing in the presence of glass beads. In a second alternate protocol, growing cells are frozen immediately in liquid nitrogen and then lysed by grinding in an industrial-strength blender in the presence of liquid nitrogen.

Equivalent mutations in the two repeats of yeast TATA-binding protein confer distinct TATA recognition specificities.

To investigate the process of TATA box recognition by the TATA box-binding protein (TBP), we have performed a detailed genetic and biochemical analysis of two Saccharomyces cerevisiae TBP mutants with altered DNA-binding specificity. The mutant proteins have amino acid substitutions (Leu-205 to Phe and Leu-114 to Phe) at equivalent positions within the two repeats of TBP that are involved in TATA element binding. In an in vivo assay that employs a nearly complete set of single point mutations of the consensus TATAAA sequence, one of the TBP mutants (TBP-L114F) recognizes the sequence TATAAG, while the other TBP mutant (TBP-L205F) recognizes one substitution at the first position of the TATA element, CATAAA, and three substitutions at the 3' end of the TATA box. Specificity patterns determined from in vitro transcription experiments with purified recombinant wild-type TBP and TBP-L205F agree closely with those observed in vivo, indicating that altered TATA utilization in the mutant strains is a direct consequence of altered TATA recognition by the mutant TBPs. The distinct TATA recognition patterns exhibited by TBP-L114F and TBP-L205F strongly suggest that in vivo, TBP binds to the TATA element in a specific orientation. The orientation predicted from these studies is further supported by the identification of intragenic suppressors that correct the defect of TBP-L205F. This orientation is consistent with that observed in vitro by crystallographic analyses of TBP-TATA box complexes. Finally, the importance of altered DNA-binding specificity in transcriptional regulation at the S. cerevisiae his4-912 delta promoter was addressed for TBP-L205F. A mutational analysis of this promoter region demonstrates that the nonconsensus TATA element CATAAA is required for a transcriptional effect of TBP-L205F in vivo. This finding suggests that the interaction of TBP with nonconsensus TATA elements may play an important regulatory role in transcription initiation.

Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides.

VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

TATA-binding protein activates transcription when upstream of a GCN4-binding site in a novel yeast promoter.

In the gal-his3 hybrid promoter, his3-GG1, GCN4 stimulates transcription at the position normally occupied by a TATA element. This expression requires two elements within gal1-10 sequences, a REB1-binding site and a second element, Z, which resides 20 base pairs upstream of the GCN4-binding site. No obvious TATA element is present in this promoter. To characterize the function of Z, we replaced it with short random oligonucleotides and selected for expression in vivo. Fourteen elements were identified and classified into groups based upon sequence and phenotypic similarities. Group 1 elements contained functional TATA sequences that were essential for activity. TATA elements can thus function when positioned upstream of a GCN4-binding site. The Group 2 elements activated transcription poorly when used as conventional TATA elements; however, mutational analyses demonstrated that their activity required TATA-like sequences. These TATA-like sequences bound the yeast TATA-binding protein (TBP) poorly in vitro but function in vivo as TBP interaction sites based upon two criteria. First mutations that improved their TATA character correspondingly improved function and second their activity could be enhanced in the presence of an altered binding specificity mutant of TBP. Furthermore, the Group 2 elements enabled the identification of mutations outside of the TATA-like core that contribute to transcriptional activation without adversely affecting TBP binding. The finding that low affinity TBP-binding sites can be used at unconventional positions suggests that many "TATA-less" promoters contain a cryptic interaction site for TBP.

Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA.

Protein-DNA recognition is often mediated by a small domain containing a recognizable structural motif, such as the helix-turn-helix or the zinc-finger. These motifs are compact structures that dock against the DNA double helix. Another DNA recognition motif, found in a highly conserved family of eukaryotic transcription factors including C/EPB, Fos, Jun and CREB, consists of a coiled-coil dimerization element the leucine-zipper and an adjoining basic region which mediates DNA binding. Here we describe circular dichroism and 1H-NMR spectroscopic studies of another family member, the yeast transcriptional activator GCN4. The 58-residue DNA-binding domain of GCN4, GCN4-p, exhibits a concentration-dependent alpha-helical transition, in accord with previous studies of the dimerization properties of an isolated leucine-zipper peptide. The GCN4-p dimer is approximately 70% helical at 25 degrees C, implying that the basic region adjacent to the leucine zipper is largely unstructured in the absence of DNA. Strikingly, addition of DNA containing a GCN4 binding site (AP-1 site) increases the alpha-helix content of GNC4-p to at least 95%. Thus, the basic region acquires substantial alpha-helical structure when it binds to DNA. A similar folding transition is observed on GCN4-p binding to the related ATF/CREB site, which contains an additional central base pair. The accommodation of DNA target sites of different lengths clearly requires some flexibility in the GCN4 binding domain, despite its high alpha-helix content. Our results indicate that the GCN4 basic region is significantly unfolded at 25 degrees C and that its folded, alpha-helical conformation is stabilized by binding to DNA.

Yeast and human TATA-binding proteins have nearly identical DNA sequence requirements for transcription in vitro.

We have analyzed the DNA sequence requirements for TATA element function by assaying the transcriptional activities of 25 promoters, including those representing each of the 18 single-point mutants of the consensus sequence TATAAA, in a reconstituted in vitro system that depends on the TATA element-binding factor TFIID. Interestingly, yeast TFIID and HeLa cell TFIID were virtually identical in terms of their relative activities on this set of promoters. Of the mutated elements, only two had undetectable activity; the rest had activities ranging from 2 to 75% of the activity of the consensus element, which was the most active. In addition, mutations of the nucleotide following the TATAAA core strongly influenced transcriptional activity, although with somewhat different effects on yeast and HeLa TFIID. The activities of all these promoters depended upon TFIID, and the level of TFIID-dependent transcription in vitro correlated strongly with their activities in yeast cells. This suggests that the in vivo activities of these elements reflect their ability to functionally interact with a single TATA-binding factor. However, some elements with similar activities in vitro supported very different levels of transcriptional activation by GAL4 protein in vivo. These results extend the degree of evolutionary conservation between yeast and mammalian TFIID and are useful for predicting the level of TATA element function from the primary sequence.

A wide variety of DNA sequences can functionally replace a yeast TATA element for transcriptional activation.

We created a library of DNA molecules in which the required TATA element of a yeast gal-his3 promoter is replaced by random-sequence oligomers averaging 16 bp in length. Surprisingly, 1% of such random sequences functionally replace the native yeast TATA element. In many cases, sequences completely unrelated to the consensus TATA element (TATAAA) stimulate transcription with equal or increased efficiency. Transcription mediated by these synthetic elements requires GAL4 and is initiated from normal his3 initiation sites, suggesting that it occurs by a mechanism indistinguishable from that involving wild-type TATA elements. Many, but not all, of these elements act as substrates for yeast TFIID in reconstituted transcription reactions in vitro. These observations indicate that yeast TFIID can stimulate transcription from TATA elements whose sequences differ from the consensus, and they suggest the possibility of alternative factors that may provide a related function for transcriptional activation.

In vitro replication of DNA containing either the SV40 or the polyoma origin.

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro. Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo. The host-cell source of DNA polymerase alpha - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase alpha - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).

Replication of simian virus 40 origin-containing DNA in vitro with purified proteins.

Simian virus 40 (SV40) DNA replication dependent on the SV40 origin of replication and the SV40 large tumor (T) antigen has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, these components included the DNA polymerase alpha-primase complex, topoisomerase I, and a fraction that contained a single-stranded DNA binding protein. The latter protein, which sediments at 5.1 S on glycerol gradients and copurifies with two major protein species of 72 and 76 kDa, was isolated solely by its ability to support SV40 DNA replication. The purified system retained the species-specific DNA polymerase alpha-primase requirement previously observed with crude fractions; the complex from HeLa cells supported SV40 replication, whereas that from calf thymus and mouse cells did not. DNA containing the polyomavirus origin of replication was replicated in a system containing polyomavirus T antigen, the HeLa single-stranded DNA binding protein-containing fraction, and DNA polymerase alpha-primase complex from mouse, but not HeLa, cells. While crude fractions yielded closed circular duplex DNA, none was detected with the purified system. Nevertheless, the addition of a crude fraction to the purified system yielded closed circular monomer products.

Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication.

The simian virus 40 (SV40) large T antigen (large tumor antigen), in conjunction with a topoisomerase, a DNA binding protein, and ATP, catalyzed the conversion of a circular duplex DNA molecule containing the SV40 origin of replication to a form with unusual electrophoretic mobility that we have named form U. Analysis of this molecule revealed it to be a highly underwound covalently closed circle. DNA unwinding was not detected with DNA containing a SV40 T-antigen binding site II mutation that renders the DNA inactive in replication. The unwinding reaction requires the action of a helicase, and SV40 T-antigen preparations contain such an activity. The T-antigen-associated ability to unwind DNA copurified with other activities intrinsic to T antigen [ability to support replication of SV40 DNA containing the SV40 origin, poly(dT)-stimulated ATPase activity, and DNA helicase]. However, in contrast to the unwinding activity, the SV40 T-antigen-associated helicase activity was not sequence-specific. A variety of labeled oligonucleotides hybridized with circular single-stranded DNA were displaced by T antigen in the presence of ATP.

Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains.

We have evidence for the formation of a stable preelongation complex during the replication of simian virus 40 (SV40) origin containing DNA (ori+ DNA) in vitro. Preincubation of ori+ DNA with HeLa cytosolic extracts and SV40-encoded large tumor antigen (T antigen) in the absence of deoxynucleoside triphosphates eliminates a lag that normally precedes replication. This effect requires ATP and is inhibited by RNase A; subsequent elongation is inhibited by aphidicolin but not by RNase A. A T antigen and SV40 origin-dependent complex can be isolated by gel-filtration chromatography of preincubation reaction mixtures. In both cases, the products formed by replication after complex formation resemble those formed during in vitro replication reactions described previously. HeLa cytosolic extract was separated into two ammonium sulfate fractions: a 0-40% fraction (AS 40) that shows low levels of DNA synthesis and a 40-65% fraction (AS 65) that is inactive by itself but stimulates synthesis when added to the AS 40 fraction. DNA synthesis by these combined fractions has the same requirements as crude extract, occurs in two stages as described above, and is sensitive to RNase A. Pretreatment of both fractions with micrococcal nuclease eliminated replication activity, whereas the combination of a pretreated fraction (either AS 40 or 65) with an untreated fraction was active. A heat-inactivated (55 degrees C, 5 min) AS 65 fraction restored replication activity to the combination of micrococcal nuclease-treated AS 40 and AS 65 fractions.

Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro.

The role of DNA polymerase alpha (pol alpha) and DNA primase has been investigated in the simian virus 40 (SV40) DNA replication system in vitro. Removal of pol alpha and primase activities from crude extracts of HeLa cells or monkey cells by use of an anti-pol alpha immunoaffinity column resulted in the loss of replication activity. The addition of purified pol alpha-primase complex isolated from HeLa cells or monkey cells restored the replication activity of depleted extracts. In contrast, the pol alpha-primase complex isolated from either mouse cells or calf thumus did not. Extracts prepared from mouse cells (a source that does not support replication of SV40) did not replicate SV40 DNA. However, the addition of purified pol alpha-primase complex isolated from HeLa cells activated mouse cell extracts. pol alpha and primase from HeLa cells were extensively purified and separated by a one-step immunoaffinity adsorption and elution procedure. Both activities were required to restore DNA synthesis; the addition of pol alpha or primase alone supported replication poorly. Crude extracts of HeLa cells that were active in SV40 replication catalyzed the synthesis of full-length linear double-stranded (RFIII) DNA in reaction mixtures containing poly(dT)-tailed pBR322 RFIII. Maximal activity was dependent on the addition of oligo(dA), ATP, and creatine phosphate and was totally inhibited by aphidicolin. Since pol alpha alone could not replicate this substrate and since there was no degradation of input DNA, we propose that other enzymatic activities associate with pol alpha, displace the non-template strand, and allow the enzyme to replicate through duplex regions.

Proteins tightly associated with the termini of replicative form DNA of Kilham rat virus, an autonomous parvovirus.

Revie et al. [Revie, D., Tseng, B. Y., Grafstrom, R. H. & Goulian, M. (1979) Proc. Natl. Acad. Sci. USA 76, 5539-5543] have proposed that the double-stranded replicative form (RF) DNA of the autonomous rodent parvovirus H-1 has protein of 60 kDa covalently bound at its 5' termini. We present evidence that the RF DNA of a similar rodent parvovirus, Kilham rat virus (KRV), also has covalently bound protein. NaDodSO4/polyacrylamide gel electrophoresis of purified, 125I-labeled RF DNA shows that proteins of 68-72, 66, 64, and 55 kDa copurify with the DNA during velocity and equilibrium sedimentation in the presence of detergents and 4 M guanidine HCl. Phenol extraction in the presence of 2-mercaptoethanol removes the 68- to 72-kDa proteins, but the 66-, 64-, and 55-kDa proteins remain tightly, but noncovalently, bound. The latter polypeptides also appear to associate with protease-treated RF DNA when mixed with uninfected cell extract. Following removal of these proteins by electrophoresis in NaDodSO4/agarose gels, two proteins (called RF TP-90 and RF TP-40), of about 90 and 40 kDa, become evident. These remain bound to the DNA and are released only after nuclease digestion of the DNA. These two proteins, apparently not of viral origin, are associated with terminal restriction fragments of the RF DNA and appear to be covalently bound to the 5' termini of both strands.

In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site.

Extracts (0.2 M NaCl) of HeLa cells support replication of DNA containing simian virus 40 (SV40) origin in the presence of SV40 large tumor (T) antigen. The reaction leads to the accumulation of high molecular weight products that represent DNA containing one parental strand and one progeny strand as well as duplex molecules that contain both strands derived from the input deoxynucleoside triphosphates. The replication reaction is inhibited by aphidicolin and by camptothecin, two inhibitors known to inhibit eukaryotic DNA replication in vivo.

Structure of the capsid of Kilham rat virus from small-angle neutron scattering.

The structure of empty capsids of Kilham rat virus, an autonomous parvovirus with icosahedral symmetry, was investigated by small-angle neutron scattering. From the forward scatter, the molecular weight was determined to be 4.0 X 10(6), and from the Guinier region, the radius of gyration was found to be 105 A in D2O and 104 A in H2O. On the basis of the capsid molecular weight and the molecular weights and relative abundances of the capsid proteins, we propose that the capsid has a triangulation number of 1. Extended scattering curves and mathematical modeling revealed that the capsid consists of two shells of protein, the inner shell extending from 58 to 91 A in D2O and from 50 to 91 A in H2O and containing 11% of the capsid scattering mass, and the outer shell extending to 121 A in H2O and D2O. The inner shell appears to have a higher content of basic amino acids than the outer shell, based on its lower scattering density in D2O than in H2O. We propose that all three capsid proteins contribute to the inner shell and that this basic region serves DNA binding and partial charge neutralization functions.

Mechanism of suppression in Drosophila: evidence for a macromolecule produced by the su(s)+ locus that inhibits sepiapterin synthase.

Genetic suppression was studied in the purple mutant of Drosophila melanogaster and in suppressed purple by measurement of sepiapterin synthase activity. The addition of ammonium sulfate fractions from adult Drosophila that contain one, two, three or four doses of su(s)+ to the suppressed purple sepiapterin synthase resulted in an inhibition that increased progressively as the dosage of su(s)+ increased; the wild-type sepiapterin synthase was not inhibited. This inhibition is caused by a heat-labile macromolecule. We suggest that the mechanism of suppression is neither transcriptional nor translational but is the result of decreased amounts, or altered properties, of the normal product of the su(s)+ locus when su(s)+ is replaced by su(s)2 or su(s)e6.