Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock -factor 32 encoded by the rpoH gene. Increased 32 levels result from both enhanced synthesis and stabilization. Previous work indicated that 32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of 32 synthesis further, we analyzed expression of rpoH-lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA-30S ribosome-tRNA f Met complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator.

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The solution structure of the central domain of the human nucleotide excision repair protein XPA, which binds to damaged DNA and replication protein A (RPA), was determined by nuclear magnetic resonance (NMR) spectroscopy. The central domain consists of a zinc-containing subdomain and a C-terminal subdomain. The zinc-containing subdomain has a compact globular structure and is distinct from the zinc-fingers found in transcription factors. The C-terminal subdomain folds into a novel alpha/beta structure with a positively charged superficial cleft. From the NMR spectra of the complexes, DNA and RPA binding surfaces are suggested.

It has proved possible to study the specificity of hydrogen bonding between the purine and pyrimidine constituents of nucleic acids by preparing derivatives which allow their interaction to be observed in a nonaqueous solvent. The nonaqueous solvent avoids the stacking interactions of the purines and pyrimidines and since the derivatives are uncharged, interionic forces are absent. In addition the interactions are not modified by the stereochemical constraints which are present when nucleotides are arranged in a polynucleotide chain. Thus these studies allow us to isolate the hydrogen bonding interactions from other factors which are important in polynucleotides. The initial infrared observations'-3 showed that uracil and adenine derivatives form hydrogen bonds with each other preferentially as compared to the extent to which they hydrogen-bond with themselves. These observations have been extended to guanine and cytosine derivatives with both infrared and nuclear magnetic resonance techniques.4-7 The latter studies show that the guanine and cytosine residues likewise form hydrogen-bonded dimers with each other much more readily than with themselves. Significantly, the affinity of adenine for uracil (or thymine) derivatives and of guanine for cytosine derivatives is highly selective; guanine or cytosine derivatives do not preferentially associate with adenine or uracil derivatives. Thus the hydrogen-bonding interactions of these molecules show a type of electronic complementarity which matches exactly the geometric complementarity that plays such an important role in our understanding of the structure of the double-stranded helical nucleic acid molecules.In the present paper we report the results of a quantitative measurement of the association constants of seven uracil derivatives and six adenine derivatives. It is found that some substituents enhance the hydrogen-bonding affinity of the adenine-uracil association while others markedly decrease it. Some of the derivatives belong to the minor bases of transfer RNA; thus these studies allow us to measure their hydrogen-bonding affinities. Other derivatives allow us to choose between the two possible sites on adenine which can be involved in hydrogen bonding. In addition it is shown that those derivatives which enhance the association of adenine and uracil with each other nonetheless do not interact with guanine or cytosine derivatives. Materials and Methods.-As in the previous study,', I derivatives of adenine with an ethyl group on Ng and of uracil with cyclohexyl on N1 were used. Seven types of uracil derivatives were studied: uracil, thymine, 3-methyluracil, 5,6-dihydrouracil, 5-bromouracil, 5-iodouracil, and 4-thiouracil. Six adenine analogues were studied: 6-aminopurine (adenine), 6-methylaminopurine, 6-dimethylaminopurine, 2-aminopurine; 2,6-diaminopurine, and 6-amino-8-bromopurine. These were purchased from the Cyclo Chemical Co., Los Angeles. Frequently they will be referred to without mentioning the 1-cyclohexyl or 9-ethyl substituents which are used to ...