Christopher J. Brandl

The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted α Helices: Crystal structure of the protein-DNA complex

The yeast transcriptional activator GCN4 is 1 of over 30 identified eukaryotic proteins containing the basic region leucine zipper (bZIP) DNA-binding motif. We have determined the crystal structure of the GCN4 bZIP element complexed with DNA at 2.9 A resolution. The bZIP dimer is a pair of continuous alpha helices that form a parallel coiled coil over their carboxy-terminal 30 residues and gradually diverge toward their amino termini to pass through the major groove of the DNA-binding site. The coiled-coil dimerization interface is oriented almost perpendicular to the DNA axis, giving the complex the appearance of the letter T. There are no kinks or sharp bends in either bZIP monomer. Numerous contacts to DNA bases and phosphate oxygens are made by basic region residues that are conserved in the bZIP protein family. The details of the bZIP dimer interaction with DNA can explain recognition of the AP-1 site by the GCN4 protein.

Two Ca2+ ATPase genes: Homologies and mechanistic implications of deduced amino acid sequences

Rabbit genomic DNA contains two genes that encode Ca2+ ATPases of fast twitch and of slow twitch (and cardiac) sarcoplasmic reticulum, respectively. The deduced amino acid sequences of the products of the two genes are highly conserved in putative Ca2+ binding regions, in sectors leading from cytoplasmic domains into transmembrane domains, and in transmembrane helices. A transport mechanism is proposed in which Ca2+ binds to negatively charged groups on amphipathic stalk sectors, becoming occluded during enzyme phosphorylation by bound ATP. Rotation of the stalk sectors is induced as the energy in the phosphorylated enzyme (E1P) is utilized in conformational changes leading to the low energy form, E2P. Rotation leads to disruption of high affinity Ca2+ binding sites and release of Ca2+ into a charge-lined membrane channel. Ca2+ then traverses the membrane by exchange diffusion.

Fast-twitch and slow-twitch/cardiac Ca2+ ATPase genes map to human chromosomes 16 and 12.

The fast-twitch and slow-twitch/cardiac Ca2+ATPase genes have been assigned to human chromosomes 16 and 12, respectively, using rodent-human somatic cell hybrids and filter hybridization analysis of cell hybrid DNA. A rabbit cDNA for the fast-twitch ATPase hybridizes to a prominent single fragment in human genomic DNA digested with the restriction enzyme BamHI. By correlating the presence of this fragment in somatic cell hybrid DNA with the human chromosome content of the hybrids, the fast-twitch ATPase gene can be assigned to human chromosome 16. A slow-twitch/cardiac ATPase cDNA clone was isolated from a human muscle cDNA library and used to detect human fragments in EcoRI-digested somatic cell hybrid DNA. By correlating the presence of these fragments with the human chromosome content of the hybrids, the slow-twitch/cardiac ATPase gene can be assigned to human chromosome 12. Thus, the two ATPase genes, which are probably related to each other by an ancient duplication event, are not syntenic in the human genome.

[22] cDNA cloning of sarcoplasmic reticulum proteins

Publisher Summary This chapter describes optimal conditions for the rapid construction of muscle cDNA libraries containing full-length transcripts. Recombinant DNA technology is a powerful tool for obtaining information on primary structures and developmental alterations in expression of specific proteins. This is especially true for intractable membrane proteins, cDNAs encoding slow-twitch/cardiac and fast-twitch forms of the Ca 2+ -ATPase, and a fast-twitch form of calsequestrin have been cloned; and studies have been initiated on the structure and developmental pattern of these proteins of the sarcoplasmic reticulum. Conditions for synthetic oligonucleotide screening and priming of cDNA synthesis are also described. As RNase is a ubiquitous molecule, extensive precautions must be taken in the preparation and handling of materials and reagents used for isolation of RNA, and for first-strand cDNA synthesis. Where possible, sterile plastic ware is used and when this is not suitable, the use of oven-baked glassware (180°, 2 hr) is recommended. To minimize handling, reagents for solutions are transferred directly or with a baked spatula to preweighed sterile tubes.

The Ca2+ ATPase of Cardiac Muscle Sarcoplasmic Reticulum

The sarcoplasmic reticulum of cardiac muscle is an internal membrane system which accumulates, sequesters and releases Ca2+. The ability of this system to regulate cytoplasmic Ca2+ concentrations is central to the control of muscle contraction (1). The predominant protein of the sarcoplasmic reticulum is an integral membrane protein with a molecular weight of 110,000. This protein, a high affinity Ca2+ pump, utilizes the energy of ATP hydrolysis to transport Ca2+ against a concentration gradient into the lumen of the sarcoplasmic reticulum (2–4). Cytoplasmic Ca2+ concentration are thereby lowered to a level where Ca2+ dissociates from troponin C, permitting muscle relaxation (5–7).