Асеев Леонид Викторович

Кандидат биологических наук


Научный сотрудник (Лаборатория регуляторной транскриптомики)

Тел.: +7 (495) 3306329

Эл. почта: leroymail@gmail.com

Образование

Период обученияСтрана, городУчебное заведениеДополнительная информация
2018 Россия, Москва Московский государственный университет имени М.В.Ломоносова Диплом специалиста по специальности Биохимия
2018 Россия, Москва Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова РАН Диплом кандидата науку по специальности Молекулярная биология

Научные интересы

Регуляция биосинтеза рибосомных белков у бактерий.

Избранные публикации

  1. Aseev L.V., Koledinskaya L.S., Boni I.V. (2016). Regulation of Ribosomal Protein Operons rplM-rpsI, rpmB-rpmG, and rplU-rpmA at the Transcriptional and Translational Levels. J. Bacteriol. 198 (18), 2494–502 [+]

    It is widely assumed that in the best-characterized model bacterium Escherichia coli, transcription units encoding ribosomal proteins (r-proteins) and regulation of their expression have been already well defined. However, transcription start sites for several E. coli r-protein operons have been established only very recently, so that information concerning the regulation of these operons at the transcriptional or posttranscriptional level is still missing. This paper describes for the first time the in vivo regulation of three r-protein operons, rplM-rpsI, rpmB-rpmG, and rplU-rpmA The results demonstrate that transcription of all three operons is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation, in parallel with the rRNA operons. By using single-copy translational fusions with the chromosomal lacZ gene, we show here that at the translation level only one of these operons, rplM-rpsI, is regulated by the mechanism of autogenous repression involving the 5' untranslated region (UTR) of the operon mRNA, while rpmB-rpmG and rplU-rpmA are not subject to this type of regulation. This may imply that translational feedback control is not a general rule for modulating the expression of E. coli r-protein operons. Finally, we report that L13, a primary protein in 50S ribosomal subunit assembly, serves as a repressor of rplM-rpsI expression in vivo, acting at a target within the rplM translation initiation region. Thus, L13 represents a novel example of regulatory r-proteins in bacteria.

    ID:1706
  2. Aseev L.V., Bylinkina N.S., Boni I.V. (2015). Regulation of the rplY gene encoding 5S rRNA binding protein L25 in Escherichia coli and related bacteria. RNA 21 (5), 851–61 [+]

    Ribosomal protein (r-protein) L25 is one of the three r-proteins (L25, L5, L18) that interact with 5S rRNA in eubacteria. Specific binding of L25 with a certain domain of 5S r-RNA, a so-called loop E, has been studied in detail, but information about regulation of L25 synthesis has remained totally lacking. In contrast to the rplE (L5) and rplR (L18) genes that belong to the polycistronic spc-operon and are regulated at the translation level by r-protein S8, the rplY (L25) gene forms an independent transcription unit. The main goal of this work was to study the regulation of the rplY expression in vivo. We show that the rplY promoter is down-regulated by ppGpp and its cofactor DksA in response to amino acid starvation. At the level of translation, the rplY expression is subjected to the negative feedback control. The 5'-untranslated region of the rplY mRNA comprises specific sequence/structure features, including an atypical SD-like sequence, which are highly conserved in a subset of gamma-proteobacterial families. Despite the lack of a canonical SD element, the rplY'-'lacZ single-copy reporter showed unusually high translation efficiency. Expression of the rplY gene in trans decreased the translation yield, indicating the mechanism of autogenous repression. Site-directed mutagenesis of the rplY 5' UTR revealed an important role of the conserved elements in the translation control. Thus, the rplY expression regulation represents one more example of regulatory pathways that control ribosome biogenesis in Escherichia coli and related bacteria.

    ID:1287
  3. Aseev L.V., Koledinskaya L.S., Boni I.V. (2014). Dissecting the extended "-10" Escherichia coli rpsB promoter activity and regulation in vivo. Biochemistry Mosc. 79 (8), 776–84 [+]

    As we have shown previously, transcription of the rpsB-tsf operon encoding essential components of the translation machinery, a ribosomal protein S2 and an elongation factor Ts, is driven by a single promoter PrpsB, which is highly conserved among γ-proteobacteria. PrpsB belongs to the extended "-10" promoter class; it comprises a TGTG-extension upstream of the "-10" hexamer TATAAA, a suboptimal "-35" region TTGGTG, and a GC-rich discriminator GCGCGC that separates the "-10" element from the transcription start site. In this work, we examined an impact of site-directed mutations in the rpsB promoter region on expression of the reporter gene PrpsB-lacZ within the E. coli chromosome as well as promoter regulation by transcription factors ppGpp and DksA upon amino acid starvation. The results show that the transcription level largely depends on both the TGTG-extension and the TTG-element in the "-35" region, as mutations in these sequences dramatically decrease the activity of the promoter. Upon induction of amino acid starvation, the rpsB promoter is negatively regulated by ppGpp due to the presence of the GC-rich discriminator, whose substitution for the AT-rich element abolished stringent control. These and other data obtained demonstrate the necessity of a natural combination of all the conserved promoter elements for efficient and regulated transcription of the essential rpsB-tsf operon.

    ID:1286
  4. Aseev L.V., Chugunov A.O., Efremov R.G., Boni I.V. (2013). A single missense mutation in a coiled-coil domain of Escherichia coli ribosomal protein S2 confers a thermosensitive phenotype that can be suppressed by ribosomal protein S1. J. Bacteriol. 195 (1), 95–104 [+]

    Ribosomal protein S2 is an essential component of translation machinery, and its viable mutated variants conferring distinct phenotypes serve as a valuable tool in studying the role of S2 in translation regulation. One of a few available rpsB mutants, rpsB1, shows thermosensitivity and ensures enhanced expression of leaderless mRNAs. In this study, we identified the nature of the rpsB1 mutation. Sequencing of the rpsB1 allele revealed a G-to-A transition in the part of the rpsB gene which encodes a coiled-coil domain of S2. The resulting E132K substitution resides in a highly conserved site, TKKE, a so-called N-terminal capping box, at the beginning of the second alpha helix. The protruding coiled-coil domain of S2 is known to provide binding with 16S rRNA in the head of the 30S subunit and, in addition, to interact with a key mRNA binding protein, S1. Molecular dynamics simulations revealed a detrimental impact of the E132K mutation on the coiled-coil structure and thereby on the interactions between S2 and 16S rRNA, providing a clue for the thermosensitivity of the rpsB1 mutant. Using a strain producing a leaderless lacZ transcript from the chromosomal lac promoter, we demonstrated that not only the rpsB1 mutation generating S2/S1-deficient ribosomes but also the rpsA::IS10 mutation leading to partial deficiency in S1 alone increased translation efficiency of the leaderless mRNA by about 10-fold. Moderate overexpression of S1 relieved all these effects and, moreover, suppressed the thermosensitive phenotype of rpsB1, indicating the role of S1 as an extragenic suppressor of the E132K mutation.

    ID:812
  5. Асеев Л.В., Левандовская A.A., Скапцова Н.В., Бони И.В. (2009). Консервативность регуляторных элементов, контролирующих экспрессию rpsB-tsf оперона у гамма-протеобактерий. Мол. биол. 43 (1), 111–8 [+]

    Оперон rpsB-tsf у бактерий кодирует два жизненно важных компонента трансляционного аппарата - рибосомный белок (р-белок) S2 и фактор элонгации Ts. Недавно мы впервые определили положение промотора оперона и показали, что экспрессия как rpsB-, так и tsf-гена у Escherichia coli негативно регулируется белком S2 на уровне трансляции. Проведённый в данной работе филогенетический анализ выявил высокую консервативность промоторной области, а также структуры 5'-нетранслируемой области (5'-НТО) rpsB-мРНК у гамма-протеобактерий. Несмотря на различия в длине и в последовательности 5'-НТО у разных представителей этой филогенетической группы, несколько коротких участков 5'-НТО оказались универсально консервативными, что предполагает их участие в регуляции экспрессии. Филогенетические предсказания подтверждены экспериментально. Показано, что предполагаемые промоторные области rpsB-генов Yersinia pestis, Haemophilus influenzae и Pseudomonas aeruginoza способны направлять транскрипцию репортерного lacZ-гена в E.coli, а активность соответствующих 5'-НТО в трансляции подвержена аутогенной репрессии р-белком S2 in vivo.

    ID:1284
  6. Асеев Л.В., Бони И.В. (2009). Внерибосомные функции бактериальных рибосомных белков. Мол. биол. 45 (5), 805–16 [+]

    Though their primary role in a cell is to serve as integral components of protein synthesis machinery, the ribosome, many of them have functions beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with other cellular components. Extraribosomal activities of some ribosomal proteins have been observed as early as in the 1970-1980s. During the last years both a list of r-proteins-moonlighters and the repertoire of their additional functions beyond the ribosome have been greatly expanded, mainly due to newly developed techniques for dissecting RNA/DNA-protein or protein-protein interactions within functional complexes involved in various cellular processes. In this review, we surveyed information on the experimentally proven as well as on presumptive extraribosomal functions which may be performed by bacterial r-proteins in a cell.

    ID:1285
  7. Aseev L.V., Levandovskaya A.A., Tchufistova L.S., Scaptsova N.V., Boni I.V. (2008). A new regulatory circuit in ribosomal protein operons: S2-mediated control of the rpsB-tsf expression in vivo. RNA 14 (9), 1882–94 [+]

    Autogenous regulation is a general strategy of balancing ribosomal protein synthesis in bacteria. Control mechanisms have been studied in detail for most of ribosomal protein operons, except for rpsB-tsf encoding essential r-protein S2 and elongation factor Ts, where even the promoter has remained unknown. By using single-copy translational fusions with the chromosomal lacZ gene and Western-blot analysis, we demonstrate here that S2 serves as a negative regulator of both rpsB and tsf expression in vivo, acting at a single target within the rpsB 5'-untranslated region (5'-UTR). As determined by primer extension, transcription of the Escherichia coli rpsB-tsf operon starts 162 nucleotides upstream of the rpsB initiation codon at a single promoter TGTGGTATAAA belonging to the extended -10 promoter class. Both the promoter signature and the 5'-UTR structure of the rpsB gene appear to be highly conserved in gamma-proteobacteria. Deletion analysis of the rpsB 5'-UTR within rpsB'-'lacZ fusions has revealed that an operator region involved in the S2 autoregulation comprises conserved structural elements located upstream of the rpsB ribosome binding site. The S2-mediated autogenous control is impaired in rpsB mutants and, more surprisingly, in the rpsA mutant producing decreased amounts of truncated r-protein S1 (rpsAIS10), indicating that S2 might act as a repressor in cooperation with S1.

    ID:1283
  8. Komarova A.V., Chufistova L.S., Aseev L.V., Boni I.V. (2005). [An Escherichia coli strain producing a leaderless mRNA from the chromosomal lac promoter]. Bioorg. Khim. 31 (5), 557–60 [+]

    A special Escherichia coli strain capable of producing a leaderless lacZ mRNA from the chromosomal lac promoter was constructed to study the mechanism of leaderless mRNA translation. The translation efficiency of this noncanonical mRNA is very low in comparison with the canonical cellular templates, but it increases by one order of magnitude in the presence of chromosomal mutations in the genes encoding the ribosomal S1 and S2 proteins. The new strain possesses obvious advantages over the commonly used plasmid constructs (first of all, due to the constant dosage of lacZ gene in the cell) and opens possibilities for investigation of the specific conditions for leaderless mRNA translation in vivo using molecular genetic approaches.

    ID:1282