Translation initiation in eukaryotes: versatility of the scanning model.

It is generally accepted that the initiation of translation in eukaryotes involves the binding of the 40S ribosomal subunit to the capped 5' end of an mRNA and subsequent scanning of 5' UTR in search of an initiation codon. However, until recently this has remained a mere hypothesis. This review describes the novel experimental evidence in support of this classical model. Data on the participation of various factors in the eukaryotic initiation process are summarized. The sequence of initiation events is described in light of the latest experimental data. The existing physical models of scanning are presented. Special attention is paid to discussion of alternative models of eukaryotic initiation of translation. It is demonstrated that the canonical mechanism of initiation is more versatile than previously thought.

Effect of self-association on the structural organization of partially folded proteins: inactivated actin.

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.

Structure and stability of recombinant protein depend on the extra N-terminal methionine residue: S6 permutein from direct and fusion expression systems.

Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression systems, respectively. The product of direct expression contained the extra N-terminal methionine residue. The structural properties and conformational stability of these permuteins were compared using 1-D (1)H-NMR, circular dichroism, intrinsic fluorescence, differential scanning calorimetry and resistance to urea-induced unfolding. A pronounced difference in all the parameters studied has been demonstrated. This means that the structure of recombinant protein can be sensitive to peculiarities of the expression and purification procedures, leading particularly to the presence or absence of the Met at the first position in the target protein sequence.

Preparation of active tRNA gene transcripts devoid of 3'-extended products and dimers.

Significant amounts (10-30%) of 3'-extended products with one or two extra nucleotides are synthesized in the course of run-off tRNA gene transcription with T7 RNA polymerase. Denaturing polyacrylamide gel electrophoresis appeared to be insufficient to provide preparative amounts of pure correct-size transcripts. Formation of dimers by tRNA gene transcripts as side products in the course of their activation is also another obstacle in preparation of biologically active transcripts. Here, we have shown that EF-Tu affinity chromatography and/or non-denaturing electrophoresis are simple and efficient tools for isolation of highly active correct-size transcripts. Conditions for transcript activation in vitro should be carefully controlled to prevent dimer formation and obtain reliable data on tRNA transcript structure and function.

Mg2+ binding and structural stability of mature and in vitro synthesized unmodified Escherichia coli tRNAPhe.

Mature tRNAPhe from Escherichia coli and the transcript of its gene lacking modified nucleotides were compared by a variety of physical techniques. Melting experiments revealed that at a low Mg2+level the transcript was partially denatured, while the mature tRNA possessed intact tertiary interactions. Mg2+binding to both tRNAs was studied by CD and UV techniques as well as by using the Mg2+-sensitive fluorescence indicator, 8-hydroxyquinoline 5-sulfonic acid. Both tRNA forms exhibited a single strong Mg2+-binding site, its dissociation constant was 10-fold higher for the transcript. Conformational changes in response to Mg2+ addition measured by CD and UV spectrometry revealed no difference for the estimated binding cooperativity and strong differences for affinities of Mg2+-binding sites for the two tRNA forms. Conformational transitions in mature and in in vitro synthesized tRNA required the binding of two Mg2+ ions per molecule and therefore should be associated not only with a single strong binding site. The Mg2+ dependence of Stokes radii measured by gel-filtration revealed insignificant differences between the overall sizes of the two tRNA forms at physiological Mg2+ levels (>1 mM). Taken together, these results suggest that modified nucleotides stabilize tertiary interactions and increase the structure stability without affecting the mechanism of Mg2+binding and overall folding of the tRNA molecule. This conclusion is supported by the known biological activity of the E. coli tRNAPhe gene transcript.

Circularly permuted dihydrofolate reductase possesses all the properties of the molten globule state, but can resume functional tertiary structure by interaction with its ligands.

It is obvious that functional activity of a protein molecule is closely related to its structure. On the other hand, the understanding of structure-function relationship still remains one of the intriguing problems of molecular biology. There is widespread belief that mutagenesis presents a real way to solve this problem. Following this assumption, we have investigated the effect of circular permutation in dihydrofolate reductase from E. coli on protein structure and functioning. It has been shown that in the absence of ligands two circularly permuted variants of dihydrofolate reductase possess all the properties of the molten globule state. However, after addition of ligands they gain the native-like structural properties and specific activity. This means that the in vitro folding of permuted dihydrofolate reductase is terminated at the stage of the molten globule formation. Interaction of permuted protein with ligands leads to the structural adjustment and formation of active protein molecules.

Determination of protein tertiary structure class from circular dichroism spectra.

Fifty-three circular dichroism (CD) spectra consisting of the spectra of 46 native proteins, 3 denatured proteins, and one oligopeptide (the spectra of two denatured proteins and oligopeptide were taken at two different temperatures) were investigated in order to examine the correlation between the shape of the CD spectrum and the tertiary structure class of the protein. Five classes were considered--all -alpha, all -beta, alpha+beta, alpha/beta, and denatured proteins. Spectra from 190 to 236 nm with 2 nm interval were described as points in 24-dimensional hyperspace, where coordinates were values of ellipticities at fixed wavelengths. This allows the spectra to be treated as patterns and subsequently analyzed using pattern recognition algorithms. Cluster analysis, which does not need predefined information about protein structure, divides spectra into several compact groups or clusters with good correlation with tertiary structure class. To visualize these results, orthogonalization procedures were imposed on the original data set in 24-dimensional space. The new 3-dimensional coordinate system demonstrated well-separated all-beta class and denatured proteins. Regions corresponding to all -alpha and especially alpha+beta and alpha/beta proteins were not as well resolved. The following approach was then applied to the original data set to obtain an objective mathematical algorithm for the determination of a protein's tertiary structure class from its CD spectrum. Regions in 24-dimensional hyperspace corresponding to all of the tertiary structure classes were found by calculating the decision functions, or equations of hyperplanes, which separate groups of spectral patterns of different classes.(ABSTRACT TRUNCATED AT 250 WORDS)