The balanced synthesis of ribosomal proteins usually occurs on the translational level according to the feed- back mechanism, when one of the ribosomal proteins encoded by the given operon, in the case of its over-syn- thesis, acts as a repressor of translation of the mRNA of the operon [1]. Such regulation in E. coli is exemplified by the S10 operon, which contains genes of 11 ribosomal proteins (S10, L3, L4, L23, L2, S19, L22, S3, L16, L29, S17) [2] whose synthesis is regulated by the ribosomal protein L4 on both the translational and transcriptional levels [3]. In the ribosome, protein L4 binds mainly with domain I of 23S rRNA and is located within the peptidyl transferase center of the large ribosomal subparticle. It is a single-domain protein consisting of a globular part locat- ed on the 50S subparticle surface and an elongated loop containing about 50 amino acid residues (a.a.) and form- ing part of the ribosome exit tunnel [4]. On binding with rRNA, the unordered loop is structured and interacts with domains II and V of 23S rRNA, which promotes the fold- ing of rRNA during the formation of the ribosome [5-7]. Nevertheless, the loop-free mutant form of protein L4 continues to regulate expression of the S10 operon pro- teins and is included into the large ribosomal subunit [8]. The regulation of the transcription and translation of the E. coli S10 operon depends on the noncoding leader region of mRNA from the 5′-end of the first gene of ribo- somal protein S10 [9]. Regions of mRNA necessary for transcriptional and translational regulation by protein L4 are overlapping but not identical [3]. Transcription is reg- ulated as a result of an attenuation mechanism when pro- tein L4 together with the transcription factor NusA increases the RNA polymerase pause duration on the ter- mination region of the leader mRNA, which results in untimely termination [2]. As a rule, ribosomal regulatory proteins bind with the mRNA region of their operon that is homologous in structure with the specific binding site on rRNA [1]. However, the major binding site of riboso- mal protein L4 on 23S rRNA does not have obvious homology with the regulatory site [10, 11]. ISSN 0006-2979, Biochemistry (Moscow), 2014, Vol. 79, No. 1, pp. 69-76. © Pleiades Publishing, Ltd., 2014. Original Russian Text © A. O. Mikhaylina, O. S. Kostareva, A. V. Sarskikh, R. V. Fedorov, W. Piendl, M. B. Garber, S. V. Tishchenko, 2014, published in Biokhimiya, 2014, Vol. 79, No. 1, pp. 87-95. 69 * To whom correspondence should be addressed. Investigation of the Regulatory Function of Archaeal Ribosomal Protein L4 A. O. Mikhaylina 1 *, O. S. Kostareva 1 , A. V. Sarskikh 1 , R. V. Fedorov 2 , W. Piendl 3 , M. B. Garber 1 , and S. V. Tishchenko 1 1 Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; fax: +7 (495) 514-0218; E-mail: lisenok020388@mail.ru 2 Hannover Medical School, Hannover 30625, Germany; fax: +49-511-532-5550; E-mail: fedorov.roman01@googlemail.com 3 Biocenter, Division of Medical Biochemistry, Innsbruck Medical University, 6020 Innsbruck, Austria; fax: +43-512-9003-73110; E-mail: wolfgang.piendl@i-med.ac.at Received September 4, 2013 Revision received October 2, 2013 Abstract—Ribosomal protein L4 is a regulator of protein synthesis in the Escherichia coli S10 operon, which contains genes of 11 ribosomal proteins. In this work, we have investigated regulatory functions of ribosomal protein L4 of the thermophilic archaea Methanococcus jannaschii. The S10-like operon from M. jannaschii encodes not 11, but only five ribosomal proteins (L3, L4, L23, L2, S19), and the first protein is L3 instead of S10. We have shown that MjaL4 and its mutant form lacking an elongated loop specifically inhibit expression of the first gene of the S10-like operon from the same organism in a cou- pled transcription-translation system in vitro. By deletion analysis, an L4-binding regulatory site has been found on MjaL3 mRNA, and a fragment of mRNA with length of 40 nucleotides has been prepared that is necessary and sufficient for the specific interaction with the MjaL4 protein. DOI: 10.1134/S0006297914010106 Key words: ribosomal protein L4, S10-like operon of mRNA, archaea, regulation of ribosomal protein synthesis, coupled transcription–translation system in vitro