20.500.12556/RUL-171744 Pomen fenilalanina za vezavo proteina TDP-43 na male RNA Importance of phenylalanine for binding of TDP-43 protein to small RNAs TDP-43 je RNA- in DNA-vezavni protein, ki veže trans-aktivacijski odzivni element DNA. Ključen je pri regulaciji metabolizma RNA, prenosu mRNA, sintezi stresnih granul in pri drugih celičnih procesih. Običajno se TDP-43 nahaja v topni obliki, pri nekaterih nevrodegenerativnih boleznih, kot sta amiotrofična lateralna skleroza (ALS) in frontotemporalna demenca (FTD), pa se kopiči v citoplazmi v obliki netopnih inkluzijskih telesc. Protein TDP-43, ki ga uvrščamo v družino heterogenih jedrnih ribonukleoproteinov (hnRNP), je sestavljen iz N-končne domene (NTD), dveh RNA-prepoznavnih motivov RRM1 in RRM2 ter iz neurejene C-končne domene (CTD). Vsebuje tudi jedrni lokalizacijski signal (NLS), signal za prenos iz jedra (NES) in več signalov za prenos v mitohondrije. Pri vezavi proteina TDP-43 na nukleinske kisline ima pomembno vlogo motiv RRM1, ki v zaporedju prepozna ponovitve UG oz. TG, motiv RRM2 pa poveča samo specifičnost vezave. V motivu RRM1 sta bistvena dva fenilalaninska aminokislinska ostanka, ki sodelujeta v π-π interakcijah nalaganja. V CTD se nahaja še osem fenilalaninov, ki mogoče tudi pomagajo pri stabilizaciji interakcije z RNA. V diplomskem delu smo v bakterijskem ekspresijskem sistemu T7 z avtoindukcijo izrazili protein TDP-43 in njegove mutante, kjer so bili v C-končni domeni in v motivih RRM1 ter RRM2 določeni fenilalaninski aminokislinski ostanki mutirani v levcinske. Proteine smo očistili z Ni$^{2+}$-afinitetno kromatografijo. Izvedli smo tudi in vitro transkripcijo malih RNA, ki smo jih uporabili pri analizi vezave proteinov na RNA z metodo termoforeze na mikroskali (MST). TDP-43 is an RNA- and DNA-binding protein initially discovered for binding to the trans-active response element of DNA. It plays a key role in regulating RNA metabolism, mRNA transport, stress granule formation and other cellular processes. Under normal conditions, TDP-43 is found in a soluble form. However, in certain neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), it accumulates in the cytoplasm in the form of insoluble inclusion bodies. TDP-43 belongs to the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and is composed of an N-terminal domain (NTD), two RNA recognition motifs (RRM1 and RRM2), and a disordered C-terminal domain (CTD). It also contains a nuclear localization signal (NLS), a nuclear export signal (NES) and several mitochondrial localization signals. In the binding of TDP-43 to nucleic acids, the RRM1 motif has a crucial role by recognizing UG or TG repeats in the sequence, while RRM2 enhances binding specificity. The RRM1 motif contains two essential phenylalanine residues that participate in π-π stacking interactions. There are also eight phenylalanines in the CTD, which may help stabilize the interaction with RNA. We successfully expressed TDP-43 and its mutants by auto-induction in a bacterial T7 expression system, in which some phenylalanine residues in C-terminal domain and in the RRM1 and RRM2 motifs of TDP-43 were mutated to leucine. The proteins were purified using Ni$^{2+}$-affinity chromatography. We also performed in vitro transcription of small RNAs, which were used to analyze protein-RNA binding using the method of microscale thermophoresis (MST). TDP-43 izražanje proteina in vitro transkripcija termoforeza na mikroskali male RNA TDP-43 protein expression in vitro transcription microscale thermophoresis small RNAs true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2025-09-01 12:45:00 2025-09-01 12:45:10 2025-09-18 03:38:53 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25711 248780291 Kovacek_Lucija_-_Pomen_fenilalanina_za_vezavo_proteina_TDP-43_na_male_RNA.pdf Kovacek_Lucija_-_Pomen_fenilalanina_za_vezavo_proteina_TDP-43_na_male_RNA.pdf 1 990026922ED9D9B8AA9BD1843DD4472F ef3e29132048b9d52a456723ecbd2a352e0b4c37a92834e855315f769f69b942 970eafe3-8720-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=215139 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0