Utjecaj temperature i proteina StpA na transkript gena antiCas u zaštiti bakterija Escherichia coli sustavom CRISPR-Cas

master's thesis

• Title (english): The effect of the temperature and StpA protein on the antiCas transcript in the CRISPR-Cas mediated protection of Escherichia coli
• Author: Tea Knežević
• Mentor: Ivana Ivančić Baće (mentor)
• Committee member: Ivana Ivančić Baće (predsjednik povjerenstva)
• Committee member: Mirela Sertić Perić (član povjerenstva)
• Committee member: Nenad Judaš (član povjerenstva)
• Committee member: Željka Soldin (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-09-22, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Mnoge bakterije i većina arheja koriste funkcionalne sustave CRISPR-Cas u obrani protiv
strane invazivne DNA. Ekspresija komponenata sustava CRISPR-Cas u E. coli regulirana je s
četiri promotora i moguća je samo ako je transkripcijski represor H-NS inaktiviran (Δhns). S
promotora anti-Pcas se prepisuje transkript gena antiCas koji nepoznatim mehanizmom utječe
na aktivnost sustava CRISPR-Cas. Paralog proteina H-NS, protein StpA u suvišku ukida
otpornost na infekciju λvir fagom u Δhns stanicama na 30℃ tako što se ponaša kao dodatni
represor. U ovom radu istražena je mogućnost da protein StpA svojom ulogom RNA šaperona
utječe na tercijarnu strukturu transkripta antiCas u ovisnosti o temperaturi i time na nukleaznu
aktivnost proteina Cas3. Praćenjem otpornosti stanica na infekciju fagom λvir utvrđeno je da
suvišak transkripta antiCas pri 31°C inhibira aktivnost CRISPR-Cas sustava neovisno o
proteinu StpA, dok je pri suvišku proteina Cas3 na 37°C potreban RNA šaperon StpA kako bi
očuvao negativni učinak. Povećana ekspresija transkripta antiCas u mutantima s inducibilnim
genima cas uzrokuje lizu stanica i na 31°C i 37°C.

Abstract (english)

Many bacteria and most archaea use functional CRISPR-Cas systems in defense against
invading DNA. The expression of CRISPR-Cas system components in E. coli is regulated by
four promoters and is only possible if the transcriptional repressor H-NS is inactivated (Δhns).
The antiCas gene transcript is transcribed from the anti-Pcas promoter, which affects the
activity of the CRISPR-Cas system by an unknown mechanism. A paralog of H-NS protein,
StpA protein in excess abolish resistance to λvir phage infection in Δhns cells at 30℃ by acting
as an additional repressor. In this paper it was investigated whether the protein StpA, with its
role as an RNA chaperone, affects the tertiary structure of the antiCas transcript in temperature
dependent manner and consequently affects the nuclease activity of the Cas3 protein. By
monitoring the resistance to phage λvir infection, it was determined that the excess of antiCas
transcript at 31°C inhibits the activity of the CRISPR-Cas system independently of the StpA
protein, while the excess of the Cas3 protein at 37°C requires RNA chaperone StpA to preserve
the negative effect. Increased expression of antiCas transcripts in mutants with inducible genes
cas causes cell lysis at both 31°C and 37°C.

• Keywords: CRISPR-Cas; HtpG; H-NS; Cas3; E. coli
• Keywords (english): CRISPR-Cas; HtpG; H-NS; Cas3; E. coli
• Language: croatian
• URN:NBN: urn:nbn:hr:217:307365
• Study programme: Title: Integrated undergraduate and graduate university study of Biology and Chemistry Education; specializations in: Integrated undergraduate and graduate university study of Biology and Chemistry Education; Course: Integrated undergraduate and graduate university study of Biology and Chemistry Education; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra edukacije biologije i kemije (magistar/magistra edukacije biologije i kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2024-09-22
• Created on: 2023-10-25 08:55:18