Strukturne lastnosti nekodirajoče RNA OxyS, udeležene pri stresnem odzivu celice

Štih, Vesna (author), Podbevšek, Peter (mentor)

V doktorskem delu smo proučevali strukturne lastnosti sRNA OxyS ter strukturno okarakterizirali interakcijo med OxyS in njeno mRNA tarčo fhlA. OxyS je 110 nukleotidov dolga trans-kodirana sRNA v E. coli, katere transkripcijo sproži povišana koncentracija vodikovega peroksida. OxyS nadzira ekspresijo številnih genov in ima pomembno vlogo v regulaciji stresnega odziva celice. Določitve sekundarne strukture OxyS smo se lotili z NMR spektroskopijo in v prvi fazi določili strukture lasnic SL1, SL2 in SL3, nato pa smo potrdili njihovo prisotnost v strukturi celotne OxyS. Odkrili smo, da je v predvideno nestrukturirani regiji OxyS prisotna lasnica SL4. V naslednjem koraku smo s kombinacijo SAXS, de novo napovedi 3D strukture in simulacij molekulske dinamike proučili tridimenzionalno strukturo OxyS. SAXS molekulska ovojnica je razkrila obliko OxyS, ki je podaljšana in spominja na bumerang z dvema različno dolgima krakoma. S prileganjem molekulske ovojnice in 3D modelov OxyS smo pokazali, da lasnici SL1 in SL2 predstavljata daljši krak, koaksialno naloženi lasnici SL3 in SL4 pa predstavljata krajši krak molekulske ovojnice. Določili smo optimizirano družino OxyS modelov in pokazali, da lahko OxyS zavzame več konformacij, ki se nekoliko razlikujejo v relativni orientaciji štirih lasnic. Proučevali smo tudi interakcijo med OxyS in mRNA fhlA. Osredotočili smo se na dve kratki zaporedji OxyS v lasnicah SL1 in SL3, ki se bazno parita s komplementarnima zaporedjema v 5' UTR fhlA. Z uporabo NMR spektroskopije smo določili strukturo modelnih oligonukleotidov fhlA1, SL1$_{Δ17}$, fhlA41 in SL3, ki vključujejo kratka komplementarna zaporedja udeležena v interakciji OxyS-fhlA, ter potrdili tvorbo kompleksov fhlA1+SL1$_{Δ17}$ in fhlA41+SL3. Ugotovili smo, da so za intermolekularno bazno parjenje potrebni kationi Mg$^{2+}$, v prisotnosti katerih pride do delnega razvitja fhlA1 in SL3. Identificirali smo specifične nukleotide, ki so vpleteni v intermolekularno bazno parjenje in na podlagi tega pokazali, da v interakciji ne sodelujejo vsi nukleotidi iz komplementarnih zaporedij OxyS in fhlA.

• Language: Slovenian
• Keywords: -
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2024
• PID: 20.500.12556/RUL-154436
• COBISS.SI-ID: 185630467
• Publication date in RUL: 14.02.2024

Secondary language

• Language: English
• Title: Structural properties of non-coding RNA OxyS involved in cell stress response

Abstract

In this doctoral thesis we studied the structural properties of sRNA OxyS and structurally characterized the interaction between OxyS and its mRNA target fhlA. OxyS is a 110 nucleotide, stable, trans-encoded small RNA found in E. coli and is induced by an increased concentration of hydrogen peroxide. OxyS has an important regulatory role in cell stress response, affecting the expression of multiple genes. We individually studied the segments of OxyS which are predicted to adopt stem-loops SL1, SL2 and SL3 and subsequently confirmed their structural integrity in full-length OxyS. Unexpectedly, stem-loop SL4 was identified in the region that was predicted to be unstructured. In the next step we combined SAXS, de novo modelling and unbiased molecular dynamics simulations to study the three-dimensional structure of OxyS. The molecular envelope of OxyS demonstrates that OxyS adopts an extended boomerang-like structure with two arms. The superimposition of 3D models onto the molecular envelope shows that SL1 and SL2 constitute the long arm, whereas coaxially stacked SL3 and SL4 constitute the short arm of the molecular envelope. We performed optimisation of the ensemble of OxyS models and demonstrated that OxyS is able to adopt multiple conformations which slightly differ in the relative orientation of the four stem-loops. We also studied the interaction between OxyS and mRNA fhlA. We focused on two short segments of OxyS SL1 and SL3 that base pair with complementary sequences within fhlA 5' UTR region. Using NMR spectroscopy we determined the secondary structure of model oligonucleotides fhlA1, SL1$_{Δ17}$, fhlA41 and SL3, which comprise the sites of OxyS-fhlA interaction and confirmed the formation of complexes fhlA1+SL1$_{Δ17}$ and fhlA41+SL3. We determined that Mg$^{2+}$ cations were required for the intermolecular base pairing to occur and demonstrated that partial unfolding of fhlA1 and SL3 takes place in their presence. We identified the specific nucleotides involved in the intermolecular base pairing and demonstrated that the interaction does not involve all complementary nucleotides from OxyS and fhlA.

• Keywords: -