[Modification of the 5' End of mRNA Leader Sequence Alters the Set of Initiation Factors Essential for Initiation of Translation].

The abundance of noncanonical mechanisms of eukaryotic initiation of translation indicates their involvement in the regulation of protein synthesis during key events in a cell life. One of the well-known examples of a noncanonical cap-independent process is the initiation of translation of mRNA with the 5'-untranslated (leader) region of the messenger encoding for the photoprotein obelin (the obelin leader). In the present work, mRNA with the obelin leader was modified by adding 45 deoxycytidyl nucleotides and a fluorescent label to its 5'end. Formation of the 48S ribosomal initiation complexes at the start codon of the modified mRNA was studied using primer extension inhibition (toeprinting). In contrast to mRNA with the intact obelin leader, translation initiation of which strictly requires the eIF4F factor, initiation on the modified mRNA can take place in the absence of this factor, although with less efficiency. The finding thus indicates the unknown function of the eIF4F factor in the first step(s) of mRNA recognition by ribosomal subunits.

[Stimulation of the Translation of Reporter mRNA in the Presence of Another mRNA in a Cell-Free System Based on Wheat Germ Extract].

The translation of uncapped mRNAs encoding luciferase and green fluorescent protein in a cell-free translation system based on wheat germ extract has been studied. It turned out that two simultaneously translated (in one tube) different templates in a certain range of concentrations not only do not compete, but mutually enhance each other's translation. It has been shown that the synthesis of luciferase in the presence of mRNA that encodes green fluorescent protein is much more effective than in the translation of only luciferase mRNA at the same concentration. Similarly, the efficiency of the synthesis of green fluorescent protein increases in the presence of the template that encodes luciferase. It follows that the total effect of the concurrent translation of two different mRNAs exceeds the sum of the effects of the translation of each mRNA separately.

Coupling of Translation Initiation and Termination Does Not Depend on the Mode of Initiation.

Recently we described a novel phenomenon observed during eukaryotic translation in a cell-free system: the coupling of initiation and termination on different mRNA molecules. Here we show that the phenomenon does not depend on a special mode of initiation. The mRNAs with certain leader sequences known to require different determinants for successful initiation were examined. Even in a case of using the intergenic internal ribosome entry site (IRES) of cricket paralysis virus RNA as the leader sequence, while no initiation factors are required, the effect of coupling is well expressed, including trials in the presence of hippuristanol as an inhibitor of eIF4A. Thus, the effect persists in the absence of scanning and does not depend on initiator tRNA and eIF2. The results suggest that the initiation factors are not involved in the coupling mechanism.

Formation of New Polysomes on Free mRNAs in a Cell-Free Translation Systems Is Accompanied by Partial Disassembly of Previously Formed Polysomes.

A method for detection of the fluorescence-labeled mRNA in translating ribosomal complexes has been developed. It is demonstrated that in the working cell-free translation system with preformed polysomes, formation of new polysomes on free mRNA takes place. For the first time, it is shown that the process is accompanied by partial disassembly of the previously formed polysomes. This result is interpreted as an indication of the direct relationship between processes of translation termination of polysomal ribosomes and translation initiation of free mRNAs.

Internal initiation of polyuridylic acid translation in bacterial cell-free system.

The task of the present work was to answer the question: is the free 5'-end needed for effective translation of a model polyribonucleotide template - polyuridylic acid - in a bacterial (E. coli) cell-free system? For this purpose, the template activities of the original polyuridylic acid with its free 5'-end and the polyuridylic acid with blocked 5'-end were compared in the bacterial cell-free translation system. To block the 5'-end, the cytidylic oligodeoxyribonucleotide with fluorescein residue at its 5'-end and uridylic oligoribonucleotide sequence at its 3'-end, schematically described as FAM(dC)10(rU)50, was covalently attached (ligated) to the 5'-end of the template polyuridylic acid. It was shown that the efficiency of polyphenylalanine synthesis on the 5'-blocked template and on the polyuridylic acid with free 5'-end was virtually the same. It was concluded that bacterial ribosomes are capable of effectively initiating translation at the polyuridylic sequence independently of the 5'-end of template polyribonucleotide, i.e. via an internal initiation mechanism, in the absence of a Shine-Dalgarno sequence and AUG start codon.

Leader sequences of eukaryotic mRNA can be simultaneously bound to initiating 80S ribosome and 40S ribosomal subunit.

Binding of mRNA leader sequences to ribosomes was studied in conditions of a cell-free translation system based on wheat germ extract. Leader sequence of TMV mRNA (the so-called omega-RNA sequence) was able to bind simultaneously 80S ribosome and 40S ribosomal subunit. It was found that nucleotide substitutions in omega-RNA resulting in destabilization of RNA structure have no effect on the complex formation with both 80S ribosome and 40S ribosomal subunit. Leader sequence of globin mRNA is also able to form a similar joint complex. It is supposed that the ability of mRNA leader sequences to bind simultaneously 80S ribosome and 40S subunit is independent of leader nature and may reflect previously unknown eukaryotic mechanisms of translation initiation.