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.
Gene expression regulation.
Initiation of replication
Affiliated Research Units
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