Comparison of the ability of mammalian eEF1A1 and its oncogenic variant eEF1A2 to interact with actin and calmodulin

The question as to why a protein exerts oncogenic properties is answered mainly by well-established ideas that these proteins interfere with cellular signaling pathways. However, the knowledge about structural and functional peculiarities of the oncoproteins causing these effects is far from comprehensive. The 97.5% homologous tissue-specific A1 and A2 isoforms of mammalian translation elongation factor eEF1A represent an interesting model to study a difference between protein variants of a family that differ in oncogenic potential. We propose that the different oncogenic impact of A1 and A2 might be explained by differences in their ability to communicate with their respective cellular partners. Here we probed this hypothesis by studying the interaction of eEF1A with two known partners – calmodulin and actin. Indeed, an inability of the A2 isoform to interact with calmodulin is shown, while calmodulin is capable of binding A1 and interferes with its tRNA-binding and actin-bundling activities in vitro. Both A1 and A2 variants revealed actin-bundling activity; however, the form of bundles formed in the presence of A1 or A2 was distinctly different. Thus, a potential inability of A2 to be controlled by Ca2+-mediated regulatory systems is revealed

Features of 80S mammalian ribosome and its subunits

It is generally believed that basic features of ribosomal functions are universally valid, but a systematic test still stands out for higher eukaryotic 80S ribosomes. Here we report: (i) differences in tRNA and mRNA binding capabilities of eukaryotic and bacterial ribosomes and their subunits. Eukaryotic 40S subunits bind mRNA exclusively in the presence of cognate tRNA, whereas bacterial 30S do bind mRNA already in the absence of tRNA. 80S ribosomes bind mRNA efficiently in the absence of tRNA. In contrast, bacterial 70S interact with mRNA more productively in the presence rather than in the absence of tRNA. (ii) States of initiation (Pi), pre-translocation (PRE) and post-translocation (POST) of the ribosome were checked and no significant functional differences to the prokaryotic counterpart were observed including the reciprocal linkage between A and E sites. (iii) Eukaryotic ribosomes bind tetracycline with an affinity 15 times lower than that of bacterial ribosomes (Kd 30 μM and 1–2 μM, respectively). The drug does not effect enzymatic A-site occupation of 80S ribosomes in contrast to non-enzymatic tRNA binding to the A-site. Both observations explain the relative resistance of eukaryotic ribosomes to this antibiotic

The concept of RNA-assisted protein folding: the role of tRNA

We suggest that tRNA actively participates in the transfer of 3D information from mRNA to peptides - in addition to its well-known, "classical" role of translating the 3-letter RNA codes into the one letter protein code. The tRNA molecule displays a series of thermodynamically favored configurations during translation, a movement which places the codon and coded amino acids in proximity to each other and make physical contact between some amino acids and their codons possible. This specific codon-amino acid interaction of some selected amino acids is necessary for the transfer of spatial information from mRNA to coded proteins, and is known as RNA-assisted protein folding

Number of tRNA binding sites on 80 S ribosomes and their subunits.

AbstractThe ability of rabbit liver ribosomes and their subunits to form complexes with different forms of tRNAPhe (aminoacyl-, peptidyl- and deacylated) was studied using the nitrocellulose membrane filtration technique. The 80 S ribosomes were shown to have two binding sites for aminoacyl- or peptidyl-tRNA and three binding sites for deacylated tRNA. The number of tRNA binding sites on 80 S ribosomes or 40 S subunits is constant at different Mg2+ concentrations (5–20 mM). Double reciprocal or Scatchard plot analysis indicates that the binding of Ac-Phe-tRNAPhe to the ribosomal sites is a cooperative process. The third site on the 80 S ribosome is formed by its 60 S subunit, which was shown to have one codon-independent binding site specific for deacylated tRNA

Improvement of urease based biosensor characteristics using additional layers of charged polymers

NRC publication: Ye

A novel enzyme biosensor for steroidal glycoalkaloids detection based on pH-sensitive field effect transfers

NRC publication: Ye