James R. Walker

Title:	Professor
Education:	Ph.D.: 1963, University of Texas; B.S.: 1960, Northwestern State College
Postdoc.:	Princeton University
Research:	DNA replication in bacteria; gene expression regulation
Office:	ESB 411
Phone:	(512) 471-1692
E-mail:	jrw@mail.utexas.edu
Postal Address:	The University of Texas at Austin Molecular Genetics & Microbiology 1 University Station  A5000 Austin TX 78712-0162
Courses taught:	BIO 226R "General Microbiology: Microbial Cell Structure and Genetics" BIO 393 "Problems in Molecular Genetics: ATPases in Basic Cellular Processes" BIO 398T "Supervised Teaching in Microbiology"

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One of our research interests is the mechanism of DNA replication in the bacterium Escherichia coli and the relation of replication to the cell division cycle. Most of the replication proteins and the genes which encode them have been identified through the work of many labs. The important problems now are to determine how the individual proteins function in controlling initiation at the replication origin, in polymerization of daughter strands at replication forks, or in terminating and segregating chromosomes. One gene of particular interest, dnaX, encodes two proteins, both of which are components of DNA polymerase III holoenzyme, the enzyme complex which catalyzes polymerization of daughter strands.

DNA polymerase III holoenzyme consists of a core (a e q subunits) plus obligatory auxiliary factors b, t, g, d, d',c and y. It has been proposed that this enzyme functions in vivo as an asymmetric dimer to coordinate leading and lagging strand synthesis. Functions of some individual subunits have been studied in vitro. t d or g d transfers b to primed templates; (a e then bind and polymerize. b acts as a sliding clamp to tether ae to the template.

Both t and g are products of one gene, dnaX. The 71 kDa t is the full-length translational product of the 643 codon dnaX messenger RNA. The shorter g is formed from within the same reading frame when the ribosomes encounter a programmed ribosomal frameshift signal over codons 428-430. About half of the ribosomes change the reading frame by shifting back one nucleotide. After incorporating one amino acid in the new frame, a stop codon is encountered and translation ends. Thus, g is identical to the first 430 amino acids of t but ends after codon 431 which incorporates a unique residue. The frameshift signal is so strong that 50% of the ribosomes shift and the ratio of t:g produced is 1:1. Purified t is a single-strand DNA-dependent ATPase and dATPase. Although g is not an ATPase (dATPase), it binds ATP and the g d complex is an ATPase. It is not clear how these ATPase activities fit into holoenzyme functions.

Specific questions under study include the function of ATPase (dATPase) activities of t and g d in polymerization. Localized mutagenesis has altered the dnaX gene (on a plasmid vector) in the region which encodes the ATP binding regions of t and g. Purified mutant t and g will be tested to determine their defectiveness in supporting synthesis in vitro and the function of ATP hydrolysis in assembling the b clamp. A second question concerns the requirement for each t and g in vivo. Are both required or can one protein substitute for the other? Knowledge of the mechanism of forming t and g allowed the construction of a mutant dnaX allele which synthesized only t (i.e., which had eliminated the frameshift signal without altering the amino acid sequence) and another allele which synthesized only g (by deleting the 3' end of dnaX). These mutant alleles have been crossed into the chromosome, replacing the wild-type dnaX gene. These studies prove that t is essential but that g is dispensable. Therefore, t can perform all the functions of g in b clamp loading plus it has some unique, essential activity. We propose that its unique function is dimerization of holoenzyme, because t is known to dimerize core in vitro, and that the dimerization is essential to coordinate leading and lagging strand replication in vivo in cellular organisms. Mutants which lack t are unable to grow because of the failure to coordinate leading and lagging strand synthesis.

The significance of g and its role(s) in vivo are being studied by the two approaches. First, mutant alleles which synthesize only t and which are mutated in selected domains are being constructed and crossed into the chromosome; the ability of alleles which encode only g to support growth of those strains will be tested to determine what activities g has in vivo. Second, conservation of the t:g pair and the programmed frameshift signal during evolution are being studied as a test of g significance. Among four genera of Enterbacteriaceae tested, all had t:g homologs and DNA sequencing has shown that the closely related Salmonella typhimurium has perfectly conserved the programmed frameshift signal. This degree of conservation suggests that g has some useful function.

A third question involves expression of the dnaX gene. This gene is located among a group of genes involved in nucleic acid metabolism.  These include apt (an adenine salvage enzyme), recR (plasmid recombination), htpG (heat shock) and adk (adenylate kinase). Preliminary evidence shows that, although the genes have separate promoters, some of the apt transcripts extend into adk.. Additional studies will investigate the mechanisms which control expression of these genes.

A second major research interest is the control of expression of the tRNA gene, argU. This gene encodes an arginine tRNA which recognizes AGA codons. Both the tRNA and cognate codon are among the rarest tRNAs and codons [the cellular concentrations of tRNAs is correlated with frequency of cognate codon usage]. argU expression is severely inhibited by a dyad symmetry region from -3 to +25 (+1 is the transcription start point) which, because of recent finding of a protein which binds this region, probably acts as an operator. Both the operator and the putative repressor protein are under study. A second question is the relevance of tRNA concentration and codon usage frequency to controlling macromolecular synthesis and growth.

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Selected Publications
 

2004

The Journal of Bacteriology 2004 Sep; 186(17): 5899-5905.

 

2003

 

2000

Suppression of a DnaX temperature-sensitive polymerization defect by mutation in the initiation gene, dnaA, requires functional oriC.
Blinkova A., E. Gines-Candelaria, J.D. Ross, J.R. Walker.
Molecular Microbiology 36 (2000): 913-25.

ABSTRACT: Temperature sensitivity of DNA polymerization and growth, resulting from mutation of the tau and gamma subunits of Escherichia coli DNA polymerase III, are suppressed by Cs,Sx mutations of the initiator gene, dnaA. These mutations simultaneously cause defective initiation at 20 degrees C. Efficient suppression, defined as restoration of normal growth rate at 39 degrees C to essentially all the cells, depends on functional oriC. Increasing DnaA activity in a strain capable of suppression, by introducing a copy of the wild-type allele, increasing the suppressor gene dosage or introducing a seqA mutation, reversed the suppression. This suggests that the suppression mechanism depends on reduced activity of DnaACs, Sx. Models that assume that suppression results from an initiation defect or from DnaACs,Sx interaction with polymerization proteins during nascent strand synthesis are proposed.

1997

Conservation of Escherichia coli dnaXprogrammed ribosomal frameshift signal in Salmonella typhimurium.
Blinkova, A., M. F. Burkart, T. D. Owens, and J.R. Walker.
The Journal of Bacteriology 179 (1997): 4438-4442.

ABSTRACT: Escherichia coli DNA polymerase III subunits tau and gamma are produced from one gene, dnaX, by a programmed ribosomal frameshift which generates the C terminal of gamma within the tau reading frame. To help evaluate the role of the dispensable gamma, the distribution of tau and gamma homologs in several other species and the sequence of the Salmonella typhimurium dnaX were determined. All four enterobacteria tested produce tau and gamma homologs. S. typhimurium dnaX is 83% identical to E. coli dnaX, but all four components of the frameshift signal are 100% conserved.

 

1995

Mutations in E. coli dnaA which suppress a dnaX(Ts) polymerization mutation and are dominant when located in the chromosomal allele and recessive on plasmids.
Gines-Candelaria, E., A. Blinkova and J.R. Walker.
The Journal of Bacteriology 177 (1995): 705-715.

ABSTRACT:  Extragenic suppressor mutations which had the ability to suppress a dnaX2016(Ts) DNA polymerization defect and which concomitantly caused cold sensitivity have been characterized within the dnaA initiation gene. When these alleles (designated Cs, Sx) were moved into dnaX+ strains, the new mutants became cold sensitive and phenotypically were initiation defective at 20 degrees C (J.R. Walker, J.A. Ramsey, and W.G. Haldenwang, Proc. Natl. Acad. Sci. USA 79:3340-3344, 1982). Detailed localization by marker rescue and DNA sequencing are reported here. One mutation changed codon 213 from Ala to Asp, the second changed Arg-432 to Leu, and the third changed codon 435 from Thr to Lys. It is striking that two of the three spontaneous mutations occurred in codons 432 and 435; these codons are within a very highly conserved, 12-residue region (K. Skarstad and E. Boye, Biochim. Biophys. Acta 1217:111-130, 1994; W. Messer and C. Weigel, submitted for publication) which must be critical for one of the DnaA activities. The dominance of wild-type and mutant alleles in both initiation and suppression activities was studied. First, in initiation function, the wild-type allele was dominant over the Cs, Sx alleles, and this dominance was independent of location. That is, the dnaA+ allele restored growth to dnaA (Cs, Sx) strains at 20 degrees C independently of which allele was present on the plasmid. The dnaA (Cs, Sx) alleles provided initiator function at 39 degrees C and were dominant in a dnaA(Ts) host at that temperature. On the other hand, suppression was dominant when the suppressor allele was chromosomal but recessive when it was plasmid borne. Furthermore, suppression was not observed when the suppressor allele was present on a plasmid and the chromosomal dnaA was a null allele. These data suggest that the suppressor allele must be integrated into the chromosome, perhaps at the normal dnaA location. Suppression by dnaA (Cs, Sx) did not require initiation at oriC; it was observed in strains deleted of oriC and which initiated at an integrated plasmid origin.

 

1993

The Escherichia coli DNA polymerase III holoenzyme contains both products of the dnaX gene, t and g, but only t is essential.
Blinkova, A., C. Hervas, P.T. Stukenberg, R. Onrust, M.C. O'Donnell and J.R. Walker.
The Journal of Bacteriology 175 (1993): 6018-6027.

The replicative polymerase of Escherichia coli, DNA polymerase III, consists of a three-subunit core polymerase plus seven accessory subunits. Of these seven, tau and gamma are products of one replication gene, dnaX. The shorter gamma is created from within the tau reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 followed by a stop codon in the new frame. Two temperature-sensitive mutations are available in dnaX. The 2016(Ts) mutation altered both tau and gamma by changing codon 118 from glycine to aspartate; the 36(Ts) mutation affected the activity only of tau because it altered codon 601 (from glutamate to lysine). Evidence which indicates that, of these two proteins, only the longer tau is essential includes the following. (i) The 36(Ts) mutation is a temperature-sensitive lethal allele, and overproduction of wild-type gamma cannot restore its growth. (ii) An allele which produced tau only could be substituted for the wild-type chromosomal gene, but a gamma-only allele could not substitute for the wild-type dnaX in the haploid state. Thus, the shorter subunit gamma is not essential, suggesting that tau can be substitute for the usual function(s) of gamma. Consistent with these results, we found that a functional polymerase was assembled from nine pure subunits in the absence of the gamma subunit. However, the possibility that, in cells growing without gamma, proteolysis of tau to form a gamma-like product in amounts below the Western blot (immunoblot) sensitivity level cannot be excluded.

Expression of the Escherichia coli dnaX gene.
Chen, K.-S., P. Saxena, and J.R. Walker.
The Journal of Bacteriology 175 (1993): 6663-6670.

ABSTRACT: The Escherichia coli dnaX gene encodes both the tau and gamma subunits of DNA polymerase III. This gene is located immediately downstream of the adenine salvage gene apt and upstream of orf12-recR, htpG, and adk. The last three are involved in recombination, heat shock, and nucleotide biosynthesis, respectively. apt, dnaX, and orf12-recR all have separate promoters, and the first two are expressed predominantly from those separate promoters. However, use of an RNase E temperature-sensitive mutant allowed the detection of lesser amounts of polycistronic messengers extending from both the apt and dnaX promoters through htpG. Interestingly, transcription of the weak dnaX promoter is stimulated 4- to 10-fold by a sequence contained entirely within the dnaX reading frame. This region has been localized; at least a portion of the sequence (and perhaps the entire sequence) is located within a 31-bp region downstream of the dnaX promoter.

 

 

 

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