Vpliv proteinov gp6 in gp7 na aktivnost promotorjev P1 in P2 bakteriofaga GIL01

Gobec, Ana Felicita (avtor), Butala, Matej (mentor)

Bakteriofag GIL01 je temperirani fag iz virusne družine Tectiviridae, ki okužuje bakterijo Bacillus thuringiensis. Medtem, ko večina temperiranih bakteriofagov, ki v aktivni litični cikel vstopijo ob poškodbah DNA, uporablja svoj represor za vzdrževanje dormantnega lizogenega cikla, GIL01 za ta namen izkorišča bakterijski represor LexA. LexA je glavni regulator bakterijskega odziva SOS, ki se vklopi ob poškodbah DNA in celici omogoči njeno popravilo. GIL01 nosi zapis za več majhnih proteinov s predvideno regulatorno funkcijo. V tej nalogi smo se osredotočili na proteina gp6 in gp7. Protein gp6 je homolog domene, ki se nahaja na aminskem koncu proteina LexA. Protein gp6 ob vezavi na promotorsko regijo P3 omogoči prepis poznih genov faga GIL01 in aktivacijo litičnega cikla. Protein gp7 tvori kompleks z bakterijskim represorjem LexA in ojača njegovo vezavo na tarčna zaporedja DNA. Cilj magistrskega dela je bil preučiti vpliv proteinov gp6 in gp7 na promotorsko regijo P1-P2 bakteriofaga GIL01, ki nadzoruje izražanje zgodnjih genov. Z analizami promotorske aktivnosti smo dokazali, da gp6 ne aktivira prepisa iz promotorske regije P1-P2. Potrdili smo, da protein gp7 deluje kot ko-represor in kot izoliran rekombinantni protein sam ne vstopa prosto v bakterijsko celico. Rezultati nakazujejo da je za aktivacijo samostojne promotorske regije P1 potreben dodatni bakteriofagni faktor. Da bi bolje okarakterizirali vezavo med LexA in gp7, smo pripravili plazmidne konstrukte za sintezo alaninskih mutantov proteina gp7, pri čemer smo posamezno aminokislino nestrukturiranega karboksilnega konca spremenili v alanin. Rezultati drugih raziskovalcev so pokazali, da noben od mutantov ni izgubil zmožnosti interagirati s proteinom LexA.

• Jezik: Slovenski jezik
• Ključne besede: bakteriofagi, bakteriofag GIL01, Bacillus thuringiensis, proteini, protein LexA, odziv SOS, fagni protein gp7, represor litičnega cikla
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: BF - Biotehniška fakulteta
• Kraj izida: Ljubljana
• Založnik: [A. F. Gobec]
• Leto izida: 2022
• PID: 20.500.12556/RUL-138150
• UDK: 577.21:578.347
• COBISS.SI-ID: 115151875
• Datum objave v RUL: 12.07.2022

GOBEC, Ana Felicita, 2022, Vpliv proteinov gp6 in gp7 na aktivnost promotorjev P1 in P2 bakteriofaga GIL01 [na spletu]. Magistrsko delo. Ljubljana : A. F. Gobec. [Dostopano 19 maj 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=138150

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: The role of gp6 and gp7 proteins on the activity of bacteriophage GIL01 promoters P1 and P2

Izvleček

Bacteriophage GIL01 is a temperate phage of the virus family Tectiviridae that infects the bacterium Bacillus thuringiensis. While most temperate bacteriophages that enter the active lytic cycle upon DNA damage use their own repressor to maintain the dormant lysogenic cycle, GIL01 uses the bacterial repressor LexA for this purpose. LexA is the master regulator of the bacterial SOS response, which is activated in the event of DNA damage and allows the cell to repair it. GIL01 carries several small proteins with a predicted regulatory function. In this work, we focused on the gp6 and gp7 proteins. The gp6 protein is a homolog of the amino terminal domain of the LexA protein, which, after binding to the P3 promoter region, activates transcription of the late genes of phage GIL01 and activation of the lytic cycle. The gp7 protein forms a complex with the bacterial repressor LexA and enhances its binding to DNA target sequences. The aim of this master thesis was to investigate the influence of the gp6 and gp7 proteins on the P1-P2 promoter region of bacteriophage GIL01, which controls the expression of early genes. Here we show that gp6 is not required for the activation of the P1-P2 promoter region. Our data support the results that the gp7 protein is a co-repressor and, as an isolated recombinant protein, does not freely enter the bacterial cell. Our results suggest that an additional bacteriophage factor is required for the activation of the P1 promoter region. To better characterize the interaction between LexA and gp7 we prepared plasmid constructs for the synthesis of single alanine mutants of gp7. Results of other researchers show that none of the mutants carrying alanine substitution at the carboxy terminal end lost the capability to interact with LexA.

• Ključne besede: bacteriophages, bacteriophage GIL01, Bacillus thuringiensis, proteins, protein LexA, SOS response, phage protein gp7, lytic repressor