Crystals, diffracting best to around 3 A Ê , have been grown from intact large and small ribosomal subunits. The bright synchrotron radiation necessary for the collection of the higher-resolution X-ray diffraction data introduces signi®cant decay even at cryo temperatures. Nevertheless, owing to the reasonable isomorphism of the recently improved crystals of the small ribosomal subunits, reliable phases have been extracted at medium resolution (5±6 A Ê ) and an interpretable ®ve-derivative MIR map has been constructed. For the crystals of the large subunits, however, the situation is more complicated because at higher resolution (2.7±7 A Ê ) they suffer from substantial radiation sensitivity, a low level of isomorphism, instability of the longest unit-cell axis and nonisotropic mosaicity. The 8 A Ê MIR map, constructed to gain insight into this unusual system, may provide feasible reasoning for the odd combination of the properties of these crystals as well as hints for future improvement. Parallel efforts, in which electron-microscopy-reconstructed images are being exploited for molecular-replacement studies, are also discussed.
Preliminary electron density maps of the large and the small ribosomal particles from halophilic and thermophilic sources, phased by the isomorphous replacement method, have been constructed at intermediate resolution. These maps contain features comparable in size with what is expected for the corresponding particles, and their packing arrangements are in accord with the schemes obtained by ab-initio procedures as well as with the motifs observed in thin sections of the crystals by electron microscopy. To phase higher resolution data, procedures are being developed for derivatization by specific labeling of the ribosomal particles at selected locations with rather small and dense clusters. Potential binding sites are being inserted either by site directed mutagenesis or by chemical modifications to facilitate cluster binding on the surface of the halophilic large and the thermophilic small ribosomal particles, which yield the crystals diffracting to highest resolution (2.9 and 7.3 A (1 A = 0.1 nm), respectively). For this purpose, the surface of these ribosomal particles is being characterized and procedures are being developed for quantitative detachment of selected ribosomal proteins and for their incorporation into core particles. The genes of these proteins are being cloned, sequenced, mutated to introduce reactive side groups, mainly cysteines, and overexpressed. In parallel, two in situ small and stable complexes were isolated from the halophilic ribosome. Procedures for their crystal production in large quantities are currently being developed. Models, reconstructed at low resolution from crystalline arrays of ribosomes and their large subunits, are being used for initial low-resolution phasing of the X-ray amplitudes. The interpretation of these models stimulated the design and the crystallization of complexes mimicking defined functional states of a higher quality than those obtained for isolated ribosomes. These models also inspired modelling experiments according to results of functional studies, performed elsewhere, focusing on the progression of nascent proteins.
The TATA box-binding protein (TBP) is required by all three eukar~otic RNA polymerases for coiTect initiation of transcriptiOn of nbosomal, messenger, small nuclear and transfer RNAs. In the most general case, pol II transcliption of mRNA begins with TBP-mediated recognition of a TATA box located imm~di ately upstream of the transcription start site. The structure of a TBP from Arabidopsis t!wliana complexed with a fourteen base pair oligonucleotide bearing the Adenovirus major late promoter (AdMLP) TATA element has been determined at l.9A resolution. Binding of the monomeric, saddle-shaped protein induces an unprecedented conformational change in the DNA. Insertion of two pairs of phenylalanine side chains into two base steps (TpA and ApG) produces two sharp kinks at either end of the sequence TATAAAAG. Between the kinks the double helix is partially unwound and smoothly bent, approximating the widened minor groove face of the TATA element to the concave surface of the molecular saddle. More recently, the structure of a ternary complex of transcription factor IIB (TFIIB), TBP, and the Ac!MLP TATA element has been determined at 2. 7 A resolution. The Cterminal/core region of TFIIB consists of two quasi-identical helical domains, separated by a cleft that grasps TBP's acidic Cterminal stiiTup. The structure of the TBP-DNA complex is essentially unaffected by contact with the basic surface of core TFIIB, which also interacts with the phospho-ribose backbone up-and downstream of the center of the TATA element. The Nterminal domain of core TFIIB is located on the downstream surface of the ternary complex, where it could interact with RNA polymerase II and help fix the transc1iption start site. The upper surface and N-terminal stirrup ofTBP, and the remainin£ surfaces of core TFIIB are available for interactions with TBP-associatecl factors, other class II initiation factors, and transcriptional activators and coactivators. The universal cell organelles facilitating the process protein biosynthesis are nucleoprotein assemblies, the ribosomes. A typical bactelial libosome weighs over 2.3 million claltons and contains 57-73 different proteins and 3 RNA chains of about 4500 nucleoticles, arranged in two subunits of unequal size. For illuminating the detailed mechanism of the translation of the genetic code into polypeptide chains, we have initiated crystallo;;·aphic studies. ~ Diffracting crystals have been grown from ribosomes and their c?mplexes with nonlibosomal components participating in protein biOsynthesis, as well as from native, chemically modified and mutated ribosomal subunits. Those that diffract to the hi crhest resolution obtained so far for 1ibosomal crystals, 2.9 A, are ;f the large ribosomal subunits from Haloarcula malismortui. X-ray data are being collected with bright synchrotron radiation at cryogenic temperatures from flash-frozen crystals. For phasing by isomorphous replacement methods, heavy atom derivatization is being performed either by soaking crystals in solution of hete1:o_polyanions and mu...
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