Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes

Understanding regulatory mechanisms of protein synthesis in eukaryotes is essential for the accurate annotation of genome sequences. Kozak reported that the nucleotide sequence GCCGCC(A/G)CCAUGG (AUG is the initiation codon) was frequently observed in vertebrate genes and that this ‘consensus’ sequence enhanced translation initiation. However, later studies using invertebrate, fungal and plant genes reported different ‘consensus’ sequences. In this study, we conducted extensive comparative analyses of nucleotide sequences around the initiation codon by using genomic data from 47 eukaryote species including animals, fungi, plants and protists. The analyses revealed that preferred nucleotide sequences are quite diverse among different species, but differences between patterns of nucleotide bias roughly reflect the evolutionary relationships of the species. We also found strong biases of A/G at position −3, A/C at position −2 and C at position +5 that were commonly observed in all species examined. Genes with higher expression levels showed stronger signals, suggesting that these nucleotides are responsible for the regulation of translation initiation. The diversity of preferred nucleotide sequences around the initiation codon might be explained by differences in relative contributions from two distinct patterns, GCCGCCAUG and AAAAAAAUG, which implies the presence of multiple molecular mechanisms for controlling translation initiation

Renaturation of the mature subtilisin BPN' immobilized on agarose beads

AbstractWe report here another example of renaturation of subtilisin BPN′(Sbtl) by using an immobilized preparation instead of applying a digestible mutant of Streptomyces subtilisin inhibitor (SSI), a proteinaceous inhibitor of Sbtl [M. Matsubara et al. (1994) FEBS Letters 342, 193–196]. The mature Sbtl was immobilized on agarose beads employing the amino group of the protein. After thorough washing, the immobilized Sbtl was subjected to denaturation in 6 M guanidine hydrochloride (GdnHCl) at pH 2.4 for 4 h, followed by renaturation in 2 M potassium acetate at pH 6.5 for 24 h. This denaturation/renaturation cycle was repeated five times. The recovered activity of the renatured immobilized Sbtl settled at a constant level after the third denaturation/renaturation cycle, demonstrating that almost 100% renaturation was attained by use of the immobilized Sbtl. This immobilized Sbtl preparation could well be utilized for the mechanistic study of protein folding. We then found that 2 M potassium acetate was superior to 2 M potassium chloride as a refolding medium and that the ability of SSI to induce the correct shape of the mature Sbtl was lacking in several refolding media in both thermodynamic and kinetic criteria. Thus the main cause for the increase of refolding yield of Sbtl by coexistence of SSI was prevention of the autolysis of Sbtl

Thermostabilization by the Improvement of Intertrimeric Residues in Thermus thermophilus Inorganic Pyrophosphatase.

Inorganic pyrophosphatase (EC. 3.6.1.1) from Thermus thermophilus (Tth PPase)is a thermostable homohexamer of 174 amino acids，and its intertrimer interface is formed mainly by the symmetric α-helix A between subunits. Amino acids and their interactions composing intertrimer interface are different in hexameric Family I PPases，and then it was deduced that Tth PPase showed high thermostability because of stabilizing this interface by interactions of these residues. In this study，we focused on Thr138 and Ala141 residues in intertrimer interface of Tth PPase to confirm the relationship between intertrimeric residues and thermostability， and then improved their combination to His and Asp/Glu (HD or HE variant). As results，the HD variant showed the highest thermostability of enzyme activity，fluorescence spectra, and quaternary structure in the wild type Tth PPase and all variants. Especially，about 38% of hexamer and almost 40% of enzyme activity were observed in HD variant after heating even at 85℃. Therefore，we suggested that the conversion to a set of ionic His138 and Asp141 at intertrimer interface had increased the thermostability of Tth PPase，and then suppressed its thermal aggregation

Effect of Cys168 substitutions on the Thermostability and the Thermal Aggregation of Thermus thermophilus Inorganic Pyrophosphatase.

Thermus thermophilus Inorganic pyrophosphatase (Tth PPase) is comprised of homohexamer，and exhibits high thermostability. However，the thermal aggregation containing the cross-linked dimer was observed after heating above 85℃. Therefore，we focused on the sole cysteine (Cys168) in C-terminalregion，and evaluated the effects of substitutions at this position on thermostability and thermal aggregation of Tth PPase. Firstly，we prepared the four Cys168-substituted variants (C168A，L ，1，and F) by site-directed mutagenesis. Although all variants formed hexamer in native state，C168A variant exhibited the highest thermostabilities for the enzyme activity and quatemary structure in wild type and all variants，while the other variants decreased them drastically as the side chain at the 168 position was much more bulky and hydrophobic in Tth PPase. Moreover， suppression of thermal aggregation for C168A variant was observed in the ANS fluorescence experiments. Therefore，we suggest that the small volume and less hydrophobicity of side chain at 168 position may contribute to the conformational thermostability, and substitution with Ala is the most suitable for thermostabilization and suppression ofthermal aggregation of Tth PPase

Comparative analysis of the base biases at the gene terminal portions in seven eukaryote genomes

Adenine nucleotides have been found to appear preferentially in the regions after the initiation codons or before the termination codons of bacterial genes. Our previous experiments showed that AAA and AAT, the two most frequent second codons in Escherichia coli, significantly enhance translation efficiency. To determine whether such a characteristic feature of base frequencies exists in eukaryote genes, we performed a comparative analysis of the base biases at the gene terminal portions using the proteomes of seven eukaryotes. Here we show that the base appearance at the codon third positions of gene terminal regions is highly biased in eukaryote genomes, although the codon third positions are almost free from amino acid preference. The bias changes depending on its position in a gene, and is characteristic of each species. We also found that bias is most outstanding at the second codon, the codon after the initiation codon. NCN is preferred in every genome; in particular, GCG is strongly favored in human and plant genes. The presence of the bias implies that the base sequences at the second codon affect translation efficiency in eukaryotes as well as bacteria

Crystal structure of d(GCGAAAGCT) containing a parallel-stranded duplex with homo base pairs and an anti-parallel duplex with Watson–Crick base pairs

A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized in the space group I4(1)22 with the unit-cell dimensions a = b = 53.4 Å and c = 54.0 Å, and the crystal structure has been determined at 2.5 Å resolution. The four nucleotide residues CGAA of the first half of the oligomer form a parallel duplex with another half through the homo base pairs, C(2):C(2)(+) (singly-protonated between the Watson– Crick sites), G(3):G(3) (between the minor groove sites), A(4):A(4) (between the major groove sites) and A(5):A(5) (between the Watson–Crick sites). The two strands remaining in the half of the parallel duplex are split away in different directions, and they pair in an anti-parallel B-form duplex with the second half extending from a neighboring parallel duplex, so that an infinite column is formed in a head-to-tail fashion along the c-axis. It seems that a hexa-ammine cobalt cation supports such a branched and bent conformation of the oligomer. One end of the parallel duplex is stacked on the corresponding end of the adjacent parallel duplex; between them, the guanine base of the first residue is stacked on the fourth ribose of another duplex