Ribosoomide lagundamine bakterites

Väitekirja elektrooniline versioon ei sisalda publikatsiooneRibosoomid on makromolekulaarsed kompleksid, mis koosnevad kahest suurest ja ühest väikesest RNAst ja paljudest erinevatest valkudest. Ribosoomides sünteesitakse kõik valgud, mida organismis leida võib, ning aktiivsete ribsoomide konsentratsioon (ja seega sünteesi kiirus) limiteerib rakkude kasvu kiirust. Ehk teisisõnu, mida kiiremini sünteesitakse uusi ribosoome, seda kiiremini kasvab ja jaguneb ka rakk. Kuna ribosomaalse RNA süntees hõlmab ca 80% raku RNA sünteesi aktiivsusest ja ribosoomi valgud moodustavad kuni veerandi raku valgumassist on selge, et mitte ainult ribosoomide funktsioon valgusünteesil vaid ka nende metabolism on rakulises majapidamises määrava tähtsusega. Tõepoolest, juba mõnda aega on teada, et aeglaselt kasvavates bakterirakkudes tegeleb enamus raku RNA lagundamise võimekusest värskelt sünteesitud ribosomaalse RNA lagundamisega. Sellegipoolest on viimase 50 aasta vältel üldiselt usutud, et kord juba valmis tehtud ja kokku pakitud ribosoomid on äärmiselt stabiilsed ning, et neid lagundatakse vaid tugeva stressi tingimustes. Samuti on meie teadmised ribosoomide lagundamise molekulaarsetest mehhanismidest bakteris üsnagi piiratud. Käesoleva doktoritöö eesmärk on kirjeldada ribosoomide lagundamist kasvavates soolekepikese (Escherichia coli) rakkudes ja heita valgust ribsoomide lagundamise mehhanismidele, molekulaarsetele radadele ning ensüümidele, mis selles protsessis osalevad. Me avastasime üllatusega, et kuigi ribosoome tõepoolest lagundatakse kasvavates bakterirakkudes, toimub see protsess vaid rakukultuuri kasvu aeglustumise perioodil, mis eelneb statsionaarse kasvufaasi saabumisele. Meil ei õnnestunud tuvastada küpsete ribosoomide lagundamist ei ühtlase kiirusega kasvavates ega ka null-kiirusega kasvavates rakkudes. Võimalik, et ribosoomide lagundamine aitab rakke neid ette valmistades eluks statsionaarses faasis, mil ei vajata suurt valgusünteesi võimekust, küll aga vabu komponente, millest elutingimuste paranedes kiiresti uusi makromolekule tootma hakata. Lisaks leidsime, et osad (kuid mitte kõik) ribosoomi RNAd inaktiveerivad mutatsioonid viivad samuti ribsoomide lagundamisele, kuid miskipärast lagundatakse siis nii mutantseid ning inaktiivseid kui metsiktüüpi ning aktiivseid ribosoome. Jällegi viitab see, et ribsoomide lagundamise eesmärk võiks olla üldise ribosoomide konsentratsiooni alandamine rakus. Kui me lisasime ribsoomide lagundamise katsesüsteemi valgusünteesi pärssivat antibiootikumi kloramfenikool, päästsime me sellega ribosoomid lagundamisest. Seda tulemust võib tõlgendada viisil, et de novo valgusüntees on vajalik ribosoomide lagundamisprogrammi käivitamiseks rakus. Testides ribosoomide lagundamise võime osas bakteritüvesid, kus puuduvad erinevad RNAd lagundavad ensüümid, leidsime kaks ensüümi, mille puudumise korral ribosoome ei lagundatud. Neist esimene, RNaas R, lõhub RNAsid alates nende tagumisest ehk 3’ otsast ning tunneb erilist lõbu kõrge sekundaarstruktuuriga RNA-de hävitamisest. RNaas R on ka eelnevalt näidatud osalevat ribosoomide lagundamisel. Teine ensüüm on seevastu suhteliselt vähetuntud endoribonukleeas nimega YbeY, mis lõikab RNAd katki keskelt, mitte ei lagunda seda otstest. See huvitav valk on arvatud osalevat ribsoomide kokkupakkimise kvaliteedikontrollil, kus ta on vajalik kõige viimases etapis, mil tuntakse ära valgusünteesil ebaõnnestuvad ribosoomid ja suunatakse need lagundamisse. Meie katsed viitavad, et seesama valk võib valla päästa ka töökorras olevate ribosoomide lagundamise, tehes ribosoomi RNAsse esimese lõike ning tekitades sellega kaitsetu 3’ otsa, mida tunneb ära RNaas R, mis omakorda suudab ribosoomi RNA täielikult lagundadaRibosomes are macromolecular complexes that consist of two large and one small RNA and of many different small proteins. The ribosome synthesizes all cellular proteins and the concentration of active ribosomes is rate limiting for cell growth. As synthesis or ribosomal RNA encompasses 80% of cellular RNA synthesis activity and the ribosomal proteins can make up half of the cellular protein mass, it is clear that ribosomal metabolism, including ribosomal degradation, makes a worthy object of study. Nevertheless, during the past half century it has been widely believed that mature ribosomes are quite stable in the cells. The major goal of this dissertation is to describe the degradation of mature ribosomes in growing E. coli cells and to shed light on the molecular mechanism of degradation. We discovered that while mature ribosomes are indeed degraded in cells growing in batch cultures, this process is limited to the slowing of growth phase, which precedes entry into the stationary phase. We were unable to detect degradation during constant-rate growth and during early stationary phase. In addition, we found that some, but not all, ribosome-inactivating mutations in 23S rRNA and 16S rRNA led to degradation of both mutant and wild-type ribosomal RNAs. Thus, unlike in yeast, the ribosome degradation in E. coli is a general process that, once initiated, does not discriminate between active and inactive ribosomes. As ribosome degradation is inhibited by the protein synthesis inhibitor chloramphenicol, we further suggest that de novo protein synthesis might be needed for triggering the degradation program. To pinpoint the enzymes responsible for degradation we tested several strains defective for different RNases. We found two RNases, RNaseR and YbeY, whose deletion saved ribosomes from degradation. RNaseR is a well studied 3’ to 5’ exonuclease whose role in degrading heavily structured RNAs, including the rRNAs, is well established. In contrast YbeY is a potential endonuclease recently implicated in a late step ribosomal quality control, which could well be the initiating endonuclease, whose cut(s) in rRNA would present substrates for RNaseR to further scavenge into mononucleotides

RUSSIAN CAPABILITIES IN CONVENTIONAL HIGH INTENSITY WARFARE. LESSONS FROM THE 2022 INVASION OF UKRAINE

The Russian Federation is today one of the world’s major military powers. The most important component of the Russian military machine is the nuclear Strategic Forces, but conventional forces are also key actors in Russia’s defense and strategic policy. The present article will briefly describe the post-Soviet evolution of Russian conventional forces and their proficiency and effectiveness in the light of the 2022 Russo-Ukrainian War. The armed forces inherited from the Soviet Union in the 1990s faced a struggle to maintain even modest capabilities. After Boris Yeltsin handed power to Vladimir Putin in 1999, sustained attempts were made to increase their capabilities. Extensive reforms were made to improve personnel conditions and proficiency, organization and equipment. The Russian military involvement in the Syrian Civil War and in the 2014–2022 Donbas conflict seemed to indicate that substantial progress had been achieved. When Russia launched its invasion of Ukraine in February 2022, military experts expected that the reformed Russian military machine would overwhelm their outmanned and outgunned foes. Contrary to expectations, the Russians suffered decisive setbacks, and despite partial successes in Southern and Eastern Ukraine they are presently embroiled in slow, costly attritional warfare. The operations in the current conflict have evidenced serious shortcomings in organization and in combined arms tactics, suggesting that the reforms have only been partially successful. They also suggest the persistence of problems that the modern Russian armed forces inherited from their predecessors, and this may be rooted in their historical military heritage and in aspects of Russian society that go back a long way in the past. The present conflict offers an opportunity for the military organizations of the Western world to learn important lessons and readdress their defense policies

TANKS IN THE RUSSO-UKRAINIAN WAR

The Russo Ukrainian war, after the initial Russian advance following the invasion in February 2022, has turned into a stalemate characterised by grinding trench and urban warfare which in some instances has been compared to WWI. Despite the relative absence of fluid operations by massed mechanised formations, armoured vehicles and tanks in particular have been intensely employed by both opponents, resulting in heavy losses. When properly used in combination with other arms, tanks have been a key factor on the battlefield. During the first year of hostilities the Russian army often suffered from a tactical mishandling of their armoured units, leading to disproportionate losses of tanks and other AFVs compared to their opponent. The great majority of the tanks employed in Ukraine so far are upgraded late Cold War models, even if deliveries by NATO countries have introduced modern Western models into the Ukrainian arsenal. The aim of this article is to give a summary of the key characteristics of the main MBTs employed in Ukraine and to provide a brief analysis of their role and impact

Forschungs- und Technologiebericht 2001

Gemäss § 8 FOG 2000 hat das Bundesministerium für Bildung, Wissenschaft und Kultur (BMBWK) gemeinsam mit dem Bundesministerium für Verkehr, Innovation und Technologie (BMVIT) dem Nationalrat bis 1. Juni des Jahres einen Lagebericht über die aus Bundesmitteln geförderte Forschung, Technologie und Innovation in Österreich in Entsprechung des § 8 Forschungsorganisationsgesetz vorzulegen. Erstmals wird damit die bisher getrennte Berichterstattung über die Lage der Forschung (jährliche Forschungsberichte des BMBWK) und die technologische Leistungsfähigkeit Österreichs (tip-Technologieberichte 1997 und 1999) zu einem einheitlichen Dokument zusammengeführt. Damit tragen die für die Förderung der Forschung, Technologie und Innovation federführenden Ministerien BMBWK und BMVIT auch dem Umstand der wechselseitigen Verflechtung der wissenschaftlichen Grundlagenforschung einerseits und der anwendungsorientierten Technologieforschung andererseits sowie ihrer Förderung auch in Form eines einheitlichen Berichtswesens Rechnung; ein Lagebericht, der sich durch seinen analytisch-deskriptiven Stil von bisherigen Berichten unterscheidet. Die Basis dieses Lageberichts bilden Beiträge der befassten Fachministerien (neben dem BMBWK und dem BMVIT auch das BMWA), von Statistik Austria sowie vor allem umfassende Analysen des „Technologie-, Informations- und Politikberatungsprogrammes“ tip1, einer Initiative des BMVIT und des BMBWK. Schließlich wurden dankenswerterweise auch Daten vom österreichischen Universitätskuratorium zur Verfügung gestellt. Zum Erreichen des Ziels der Bundesregierung, die Forschungsquote in Österreich bis zum Jahr 2002 auf 2,0 % und bis zum Jahr 2005 auf 2,5 % des Bruttoinlandsproduktes zu erhöhen2, sind große Anstrengungen und koordinierte Initiativen der verschiedenen Akteure der Forschungs- und Technologiepolitik nötig. Aus diesem Grund ist der Informationsbedarf entsprechend hoch. Dieser Bericht leistet als Teil einer umfassenderen Berichterstattung einen Beitrag zur Deckung dieses Informationsbedarfes

Toxins MazF and MqsR cleave <i>Escherichia coli</i> rRNA precursors at multiple sites

<p>The endoribonuclease toxins of the <i>E. coli</i> toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, <i>E. coli</i> MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential RNA-sequencing to map the toxin-cleaved 5′- and 3′-ends. Our results show that the MazF and MqsR cleavage sites are located within structured rRNA regions and, therefore, are not accessible in assembled ribosomes. Most of the rRNA fragments are located in the aberrant ribosomal subunits that accumulate in response to toxin induction and contain unprocessed rRNA precursors. We did not detect MazF- or MqsR-cleaved rRNA in stationary phase bacteria and in assembled ribosomes. Thus, we conclude that MazF and MqsR cleave rRNA precursors before the ribosomes are assembled and potentially facilitate the decay of surplus rRNA transcripts during stress.</p