Structure of a yeast hypothetical protein selected by a structural genomics approach.

Yeast hypothetical protein YBL036C (SWISS-PROT P38197), initially thought to be a member of an 11-protein family, was selected for crystal structure determination since no structural or functional information was available. The structure has been determined independently by MIR and MAD methods to 2.0 A resolution. The MAD structure was determined largely through automated model building. The protein folds as a TIM barrel beginning with a long N-terminal helix, in contrast to the classic triose phosphate isomerase (TIM) structure, which begins with a beta-strand. A cofactor, pyridoxal 5'-phosphate, is covalently bound near the C-terminal end of the barrel, the usual active site in TIM-barrel folds. A single-domain monomeric molecule, this yeast protein resembles the N-terminal domain of alanine racemase or ornithine decarboxylase, both of which are two-domain dimeric proteins. The yeast protein has been shown to have amino-acid racemase activity. Although selected as a member of a protein family having no obvious relationship to proteins of known structure, the protein fold turned out to be a well known and widely distributed fold. This points to the need for a more comprehensive base of structural information and better structure-modeling tools before the goal of structure prediction from amino-acid sequences can be realised. In this case, similarity to a known structure allowed inferences to be made about the structure and function of a widely distributed protein family.

Structural genomics of enzymes involved in sterol/isoprenoid biosynthesis.

X-ray structures of two enzymes in the sterol/isoprenoid biosynthesis pathway have been determined in a structural genomics pilot study. Mevalonate-5-diphosphate decarboxylase (MDD) is a single-domain alpha/beta protein that catalyzes the last of three sequential ATP-dependent reactions which convert mevalonate to isopentenyl diphosphate. Isopentenyl disphosphate isomerase (IDI) is an alpha/beta metalloenzyme that catalyzes interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, which condense in the next step toward synthesis of sterols and a host of natural products. Homology modeling of related proteins and comparisons of the MDD and IDI structures with two other experimentally determined structures have shown that MDD is a member of the GHMP superfamily of small-molecule kinases and IDI is similar to the nudix hydrolases, which act on nucleotide diphosphatecontaining substrates. Structural models were produced for 379 proteins, encompassing a substantial fraction of both protein superfamilies. All three enzymes responsible for synthesis of isopentenyl diphosphate from mevalonate (mevalonate kinase, phosphomevalonate kinase, and MDD) share the same fold, catalyze phosphorylation of chemically similar substrates (MDD decarboxylation involves phosphorylation of mevalonate diphosphate), and seem to have evolved from a common ancestor. These structures and the structural models derived from them provide a framework for interpreting biochemical function and evolutionary relationships.

Formation of concentration gradient and its application to DNA capillary electrophoresis.

A new method to introduce the concentration gradient into the capillary has been developed and its application to DNA capillary electrophoresis is presented. The concentration gradient produced by mixing 5% w/v polyacrylamide-co-poly(N-dimethylacrylamide) (PAM-co-PDMA) solution and 1 x Tris/N-tris(hydroxymethyl)methyl-3-amino-propanesulfonic acid/EDTA (TT) + 5 M urea buffer was successfully achieved by using two programmable syringe pumps with strict control of dead volume, flow rate, and pressure balance. This method has the advantages of high stability, reproducibility, and versatility. The column with concentration gradient greatly improved the resolution, especially for the large DNA fragments, due to a decrease in band width broadening with time. A column containing 2-4% w/v gradient in four steps had a longer read length, shorter separation time and better resolution (after 380 base) than that of 4% w/v single concentration polymer solution. The number of steps in the gradient had almost no effect on the performance. The change in the average concentration by relocating the position of the same step gradient, i.e., a combination of different low concentration to high concentration polymer solution ratios, resulted in a different migration time, read length and resolution.

Structural genomics: beyond the human genome project.

With access to whole genome sequences for various organisms and imminent completion of the Human Genome Project, the entire process of discovery in molecular and cellular biology is poised to change. Massively parallel measurement strategies promise to revolutionize how we study and ultimately understand the complex biochemical circuitry responsible for controlling normal development, physiologic homeostasis and disease processes. This information explosion is also providing the foundation for an important new initiative in structural biology. We are about to embark on a program of high-throughput X-ray crystallography aimed at developing a comprehensive mechanistic understanding of normal and abnormal human and microbial physiology at the molecular level. We present the rationale for creation of a structural genomics initiative, recount the efforts of ongoing structural genomics pilot studies, and detail the lofty goals, technical challenges and pitfalls facing structural biologists.

Multi-capillary optical waveguides for DNA sequencing.

Cylindrical capillaries can be used as optical elements in a waveguide, where refraction will confine an appropriately focused light beam to pass through the interiors of successive capillaries in a flat parallel array. Such a capillary waveguide allows efficient illumination of samples in multiple capillaries with relatively little laser power. Analytical expressions derived under paraxial and thin-lens approximations provide guidance in selecting the capillary sizes and the refractive indices that will produce the waveguiding effect, but accurate predictions require exact ray tracing. Small reflective losses as the light passes through the capillary surfaces cause cumulative intensity decreases, but the resulting lack of uniformity can be compensated to a considerable extent by illuminating the capillary array from both sides. A 12-capillary waveguide illuminated from both sides in air has a difference of less than 10% from the strongest to the weakest illumination. By increasing the refractive index of both the external medium and the contents of the capillaries, a 96-capillary waveguide for DNA sequencing could be produced that would also provide nearly uniform illumination. A 12-capillary, bi-directionally illuminated waveguide system for DNA sequencing has been constructed. The two focused laser beams are delivered by integrated fiber optic transmitters (IFOTs), and fluorescence is collected by a set of optical fibers whose spacing exactly matches that of the capillaries in the waveguide. The system is easy to align and provides sensitive detection of fluorescence with minimal cross-talk between channels.

Pausing and termination by bacteriophage T7 RNA polymerase.

Two types of sites are known to cause pausing and/or termination by bacteriophage T7 RNA polymerase (RNAP). Termination at class I sites (typified by the signal found in the late region of T7 DNA, TPhi) involves the formation of a stable stem-loop structure in the nascent RNA ahead of the point of termination, and results in termination near runs of U. Class II sites, typified by a signal first identified in the cloned human preproparathyroid hormone (PTH) gene, generate no evident structure in the RNA but contain a conserved sequence ahead of the point of termination, and also contain runs of U. Termination at class I and class II sites may involve non-equivalent mechanisms, as mutants of T7 RNA polymerase have been identified that fail to recognize class II sites yet continue to recognize class I sites. In this work, we have analyzed pausing and termination at several class II sites, and variants of them. We conclude that the 7 bp sequence ATCTGTT (5' to 3' in the non-template strand) causes transcribing T7 or T3 RNA polymerase to pause. Termination 6 to 8 bp past this sequence is favored by the presence of runs of U, perhaps because they destabilize an RNA:DNA hybrid. The effects of T7 lysozyme on pausing and termination are consistent with the idea that termination involves a reversion of the polymerase from the elongation to the initiation conformation, and that lysozyme inhibits the return to the elongation conformation. A kinetic model of pausing and termination is presented that provides a consistent interpretation of our results.

Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme.

Bacteriophage T7 lysozyme is known to inhibit transcription by T7 RNA polymerase. Lysozyme present before initiation inhibited the synthesis of long RNA chains but did not inhibit elongation when added shortly after chains were initiated. A combination of gel-shift and transcription assays showed that lysozyme and polymerase form a 1:1 complex that binds promoter DNA and makes abortive transcripts, indicating that lysozyme has little effect on the early steps of transcription. Extension of stalled transcription complexes suggested that a transcribing polymerase becomes resistant to lysozyme inhibition after synthesis of an RNA chain as short as 15 nucleotides. It seems likely that bound lysozyme prevents an initiating polymerase from converting to an elongation complex. This conversion is thought to involve both a conformational change in the polymerase and the binding of nascent RNA. Gel-shift experiments indicated that lysozyme does not interfere with the binding of RNA, so it probably prevents a necessary conformational change in the polymerase. Lysozyme also increased pausing or termination at two sites in lambda DNA and at a site near the right end of the concatemer junction of T7 DNA. If pausing at these sites involves a reversal from the elongation to the initiation conformation, lysozyme may increase pausing or termination by "locking in" the initiation conformation. The arrest of transcription complexes near promoters and near the right end of the concatemer junction almost certainly must relate to lysozyme's ability to stimulate replication, maturation and packaging of T7 DNA during T7 infection.

Mutant bacteriophage T7 RNA polymerases with altered termination properties.

We have identified mutants of bacteriophage T7 RNA polymerase (RNAP) that are altered in their ability to pause or terminate at a variety of signals. These signals include a terminator found fortuitously in the human preproparathyroid hormone (PTH) gene, a pause site found in the concatamer junction (CJ) of replicating T7 DNA, and termination signals that are also utilized by Escherichia coli RNAP (e.g. rrnB T1 and T2). Whereas the mutant enzymes terminate normally at the late terminator in T7 DNA (T(phi)) and rrnB T2, they fail to terminate at one of the termination sites of rrnB T1, and also fail to recognize the PTH and CJ signals. The mutant enzymes exhibit normal processivity on linear templates, but show a slightly reduced processivity on supercoiled templates and terminate more efficiently when synthesizing poly(U) tracts. The mutant enzymes also show a decreased tendency to produce aberrant transcription products from DNA templates having protruding 3' ends. T7 lysozyme (an inhibitor of T7 RNAP) has been shown to exert its action by preventing the transition of the RNAP from an unstable initiation complex (IC) to a stable elongation complex (EC). We have found that T7 lysozyme enhances recognition of CJ by wild-type T7 RNAP, and that mutant T7 RNAPs that show increased sensitivity to lysozyme show enhanced recognition of this signal, even in the absence of lysozyme. These results, together with the observation that the mutations that result in the termination-deficient phenotype affect a region of the RNAP that has been implicated in RNA binding and upstream promoter contacts, support the hypothesis that, in some cases, termination represents a reversal of the events that occur during initiation.

Selection, identification, and genetic analysis of random mutants in the cloned primase/helicase gene of bacteriophage T7.

T7 gene 4 specifies two overlapping proteins 4A, a 566-amino acid primase/helicase, and 4B, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. The 4A' gene, which has a leucine codon replacing the 4B initiation codon, specifies a single 566-amino acid protein that can provide the primase and helicase functions required for normal T7 growth. We selected N-methyl-N'-nitro-N-nitrosoguanidine mutants in the cloned 4A' gene that no longer support the growth of a phage that completely lacks gene 4. Genetic mapping of the 76 mutations found them to be distributed throughout the protein, including both the N-terminal and C-terminal halves of the molecule thought to represent primase and helicase domains, respectively. Complementation tests with partially and completely defective phage showed that all but five of the mutants lacked helicase function but retained primase function. The other five, which lacked both functions, all made short proteins, including one missing only 60 amino acids. No mutations lacked only primase function, and none mapped within the first 105 amino acids, which includes the 63-amino acid region unique to 4A that contains elements required to recognize primase sites. Forty-six mutations were sequenced and included 27 missense mutations affecting 25 amino acids. Many mutations in the N-terminal half of the protein affected its solubility in cell extracts. Mutations in the C-terminal half clustered in or near five helicase consensus sequences. Biochemical analysis of nine of the mutant proteins is described in the accompanying paper (Washington, M. T., Rosenberg, A. H., Griffin, K., Studier, F. W., and Patel, S. S. (1996) J. Biol. Chem. 271, 26825-26834).

Biochemical analysis of mutant T7 primase/helicase proteins defective in DNA binding, nucleotide hydrolysis, and the coupling of hydrolysis with DNA unwinding.

We characterized nine helicase-deficient mutants of bacteriophage T7 helicase-primase protein (4A') prepared by random mutagenesis as reported in the accompanying paper (Rosenberg, A. H., Griffin, K., Washington, M. T., Patel, S. S., and Studier, F. W. (1996) J. Biol. Chem. 271, 26819-26824). Mutants were selected from each of the helicase-conserved motifs for detailed analysis to understand better their function. In agreement with the in vivo results, the mutants were defective in helicase activity but were active in primase function. dTTP hydrolysis, DNA binding, and hexamer formation were examined. Three classes of defective mutants were observed. Group A mutants (E348K, D424N, and S496F), defective in dTTP hydrolysis, lie in motifs 1a, 2, and 4 and are possibly involved in NTP binding/hydrolysis. Group B mutants (R487C and G488D), defective in DNA binding, lie in motif 4 and are responsible directly or indirectly for DNA binding. Group C mutants (G116D, A257T, S345F, and G451E) were not defective in any of the activities except the helicase function. These mutants, scattered throughout the protein, appear defective in coupling dTTPase activity to helicase function. Secondary structural predictions of 4A' and DnaB helicases resemble the known structures of RecA and F1-ATPase enzymes. Alignment shows a striking correlation in the positions of the amino acids that interact with NTP and DNA.

Polyacrylamide solutions for DNA sequencing by capillary electrophoresis: mesh sizes, separation and dispersion.

Two preparations of linear polyacrylamide with average molecular weights of 0.37 million and 1.14 million Da, and a deuterated preparation with an average molecular weight of 1.71 million Da, were used to study the effects of molecular weight, polydispersity, and concentration on the mesh size of entangled polymers in a DNA sequencing buffer solution and their ability to resolve DNA sequencing reactions by capillary electrophoresis. The polyacrylamide concentrations were above the overlap threshold C*, the concentration above which an entangled polymer network is expected to form. Small angle neutron scattering experiments showed that between 1% and 8% polyacrylamide, the mesh size ( xi ) can be expressed by the relation xi = 2.09C-0.76, where xi is in A and C is the polymer concentration in g/mL. The mesh size depended only on the concentration and was independent of the average molecular weight of the polyacrylamide. Consistent with this result, electrophoretic mobilities of DNA moving through the polymer network depended almost entirely on the polyacrylamide concentration and not on its molecular weight or polydispersity. Although separation was little affected, band sharpness persisted to longer DNAs when the polymer network contained a higher fraction of larger polyacrylamide molecules. We postulate a dispersive effect that depends on the size of the DNA and the resiliency of the polymer network. This interpretation provides a rationale for optimizing the design of polymer solutions to sieve DNA for sequencing by capillary electrophoresis.

Assembly of T7 capsids from independently expressed and purified head protein and scaffolding protein.

Prohead-like capsid shells containing the scaffolding and head proteins of bacteriophage T7 were isolated after both proteins were expressed from the cloned genes in the same cell. When the head-tail connector protein was also expressed, the isolated capsids contained neither connector nor scaffolding protein and resembled mature phage capsids rather than proheads. However, only a small fraction of the head protein was converted to stable capsid structures in either case. Purified scaffolding protein (expressed individually from the cloned gene) appeared to be a monomer in solution; purified head protein appeared to be a tetramer. The purified proteins reacted in the presence of polyethylene glycol or dextran to produce prohead-like capsid shells and also polycapsids consisting primarily of head protein, similar to the polycapsids observed after infection by T7 mutants lacking connector or core proteins. Neither capsids nor polycapsids were produced in the absence of scaffolding protein. Polycapsids were usually the predominant product even when scaffolding protein was in excess, and a small fraction of scaffolding protein catalyzed the conversion of an excess of head protein to polycapsids. Our results suggest that the first step in the natural pathway to prohead formation is the assembly of incomplete prohead shells, which are normally closed by insertion of a connector-core complex. In the absence of a functional connector-core complex, incomplete capsid shells apparently react further to form polycapsids or completely closed capsid shells.

Purification and characterization of T7 head-tail connectors expressed from the cloned gene.

Cloned gene 8, which specifies the protein of the head-tail connector of bacteriophage T7, was expressed in Escherichia coli. Extracts prepared in a low-salt buffer gave rise to free monomers, assembled connectors, and various complexes and aggregates. Connectors isolated as single peaks from DEAE-Sepharose and phosphocellulose chromatography gave separate peaks of monomers and stable connectors upon hydroxylapatite chromatography perhaps because of dissociation of monomer-connector complexes or disassembly of unstable connectors. Electron microscopy showed that the connectors readily formed ordered arrays after hydroxylapatite chromatography but not before. Addition of 100 mM NaCl to the buffer used to prepare extracts eliminated most complexes and aggregates and gave rise almost entirely to monomers and stable connectors that formed arrays even before hydroxylapatite chromatography. The distribution of masses determined by scanning transmission electron microscopy would be consistent with a mixed population of stable connectors containing 12 or 13 monomers, and the same preparation gave two bands upon agarose gel electrophoresis. Connectors bound linear, circular and supercoiled DNA, whereas monomers did not, as determined by a gel-shift assay. No ATPase activity was detected in either monomer or connector preparations in the absence or presence of DNA.

Isolation of transcriptionally active mutants of T7 RNA polymerase that do not support phage growth.

Mutants of bacteriophage T7 RNA polymerase defective in functions other than transcription were sought by random chemical mutagenesis of the cloned gene and selection for inability to support the growth of a T7 mutant whose growth is dependent on T7 RNA polymerase supplied by the host cell. About half of the mutant clones appeared unable to make full-length T7 RNA polymerase, many of them producing a truncated protein. Among 116 mutants expressing full-length protein, two-thirds were severely impaired in transcription, but a surprisingly high one-third were able to direct significant transcription in vivo. Both types of mutation were distributed across much of the gene, as determined by a rapid genetic mapping procedure that allows the lethal mutation in each clone to be localized. One mutation (isolated twice) allowed normal gene expression but prevented the formation of mature ends of T7 DNA from concatemers, which normally happens during packaging into phage particles. Thirty-seven of the mutations appeared to increase the sensitivity of the polymerase to inhibition by T7 lysozyme; all were suppressed by mutations in the lysozyme gene, including one suppressor constructed to retain full amidase activity but to be unable to bind T7 RNA polymerase. The two lysozyme-hypersensitive polymerase mutants analyzed in detail showed premature cessation of transcription during infection. Early proteins and those late proteins specified by genes as far right in T7 DNA as genes 8-9 appeared to be produced normally, but expression of genes farther to the right was strongly depressed. DNA replication was depressed about 50% in one of these mutants and 90% in the other, even though the T7 replication proteins were made in normal amounts at the normal time.

Ligation of hexamers on hexamer templates to produce primers for cycle sequencing or the polymerase chain reaction.

A method is described for the ordered ligation of hexanucleotides (hexamers) in solution to produce unique longer oligonucleotides. To form an 18-mer, for example, six different hexamers are selected that can base pair unambiguously to form a double-stranded complex of indefinite length. In the most efficient arrangement, each hexamer forms three complementary base pairs with two other hexamers, generating complementary chains of contiguous hexamers with strand breaks staggered by three bases. Two adjacent hexamers in one chain contain 5' phosphate groups and the others are unphosphorylated. Both T4 and T7 DNA ligase can ligate the phosphorylated hexamers to their neighbors in such a complex at hexamer concentrations in the 50-100 microM range, producing an 18-mer and leaving three unphosphorylated hexamers. Twenty-nine of 34 complexes that satisfied the requirements for unambiguous ligation generated the desired 18-mers, which could be used directly for cycle sequencing or, after removal of the unreacted hexamers, for polymerase chain reactions (PCR). Comparable ligation reactions also produced 12-, 24-, and 30-mers. With a library of all 4096 possible hexamers, unambiguous ligation has the potential to produce more than 82% of all possible 18-mers and could readily supply the oligonucleotides needed for DNA sequencing by primer walking, for PCR, or for gene synthesis.

Consecutive low-usage leucine codons block translation only when near the 5' end of a message in Escherichia coli.

Insertion of nine consecutive low-usage CUA leucine codons after codon 13 of a 313-codon test mRNA strongly inhibited its translation without apparent effect on translation of other mRNAs containing CUA codons. In contrast, nine consecutive high-usage CUG leucine codons at the same position had no apparent effect, and neither low- nor high-usage codons affected translation when inserted after codon 223 or 307. Additional experiments indicated that the strong positional effect of the low-usage codons could not be accounted for by differences in stability of the mRNAs or in stringency of selection of the correct tRNA. The positional effect could be explained if translation complexes are less stable near the beginning of a message: slow translation through low-usage codons early in the message may allow most translation complexes to dissociate before they read through.

The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase.

The lysozyme of bacteriophage T7 is a bifunctional protein that cuts amide bonds in the bacterial cell wall and binds to and inhibits transcription by T7 RNA polymerase. The structure of a mutant T7 lysozyme has been determined by x-ray crystallography and refined at 2.2-A resolution. The protein folds into an alpha/beta-sheet structure that has a prominent cleft. A zinc atom is located in the cleft, bound directly to three amino acids and, through a water molecule, to a fourth. Zinc is required for amidase activity but not for inhibition of T7 RNA polymerase. Alignment of the zinc ligands of T7 lysozyme with those of carboxypeptidase A and thermolysin suggests structural similarity among the catalytic sites for the amidase and these zinc proteases. Mutational analysis identified presumed catalytic residues for amidase activity within the cleft and a surface that appears to be the site of binding to T7 RNA polymerase. Binding of T7 RNA polymerase inhibits amidase activity.

Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system.

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages. Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNA(AGG) in the cell to become sequestered in translating peptidyl-tRNA(AGG) -mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome of frameshift, hop, or terminate translation.

DNA sequencing by primer walking with strings of contiguous hexamers.

When template DNA is saturated with a single-stranded DNA binding protein (SSB), strings of three or four contiguous hexanucleotides (hexamers) can cooperate through base-stacking interactions to prime DNA synthesis specifically from the 3' end of the string. Under the same conditions, priming by individual hexamers is suppressed. Strings of three of four hexamers representing more than 200 of the 4096 possible hexamers primed easily readable sequence ladders at more than 75 different sites in single-stranded or denatured double-stranded templates 6.4 kilobases to 40 kilobase pairs long, with a success rate of 60 to 90 percent. A synthesis of 1 micromole of hexamer supplies enough material for thousands of primings, so multiple libraries of all 4096 hexamers could be distributed at a reasonable cost. Such libraries would allow rapid and economical sequencing. Automating this strategy could increase the speed and efficiency of large-scale DNA sequencing by at least an order of magnitude.