Studies on the mechanisms of RNA polymerase II-dependent transcription elongation
Kuupäev
2011-07-19
Autorid
Ajakirja pealkiri
Ajakirja ISSN
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Kirjastaja
Abstrakt
Kõigis tuumaga rakkudes on geneetilise informatsiooni kandjad - DNA molekulid - kõvasti kokku pakitud, et tagada nende mahtumine rakutuuma. DNA lahtipakkimine on oluline mitmete rakuliste protsesside läbiviimiseks, sealhulgas transkriptsiooniks. Transkriptsiooni käigus kopeeritakse informatsioon DNA-lt mRNA-ks ning seejärel valkudeks. Seda protsessi viib läbi RNA polümeraas II, mille tööprotsess jagatakse kolmeks etapiks – initsiatsioon, elongatsioon ja terminatsioon. Käesolevas töös uuriti põhiliselt elongatsiooni käigus toimuvaid muutusi DNA-l.
Esmane DNA kokkupakkimise tase on nn nukleosoomne struktuur. Sellisel juhul on DNA seotud histoonivalkudega. On teada, et kui geeni transkriptsioonitase on kõrge, siis eemaldatakse nukleosoomid DNA-lt. Uurisime, mis määrab selle ala ulatuse, kust nukleosoomid transkriptsiooni käigus kaovad. Selgus, et DNA struktuur muutub ainult siis, kui ta on seotud elongeeriva RNA polümeraasiga ning nuklesoome ei eemaldata sealt, kuhu polümeraas ei jõua.
Eelpool mainitud nukleosoomne DNA struktuur on RNA polümeraasile suureks takistuseks. Veel suuremaks takistuseks peetakse aga DNA ja valkude kompleksi, mida nimetatakse heterokromatiiniks ehk vaigistatud kromatiiniks. Heterokromatiini puhul on DNA seotud lisaks histoonidele veel teistegi valgukompleksidega. Vaatasime, kas juba transkribeeriv polümeraas suudab heterokromatiini läbida. Saime teada, et suudab küll ning vaigistavad kompleksid eemaldatakse DNA-lt elongeeriva polümeraasi möödumisel.
Nii varasemates töödes kui ka meie katsetes ilmnes, et transkriptsiooni indutseerimisel on mudelgeenil näha rohkem RNA polümeraasi geeni alguses kui geeni lõpus. Kuna katsed olid tehtud kasutades rakkude kogu populatsiooni, tahtsime teada, kas ka ühe raku tasemel on RNA polümeraas transkribeeritaval geenil samamoodi jaotunud. Leidsime, et üksikul transkribeeritaval DNA fragmendil on polümeraasi molekulid jaotunud ühtlaselt.
Antud tööst saadud tulemused on oluliseks täienduseks baasteadmistele transkriptsiooni elongatsioonist ning rikastavad arusaama RNA polümeraas II sõltuva transkriptsiooni elongatsiooniprotsessist.
In all eukaryotic cells the carriers of genetic information - DNA molecules - are tightly packed to fit in the nucleus. Unpacking DNA is crucial for several cellular processes, including transcription. During RNA polymerase II (RNAPII)-dependent transcription one strand of DNA is used to synthesise complementary mRNA. RNA synthesis is divided into three main phases – initiation, elongation and termination. The main aim of the current thesis was to elucidate the mechanisms of elongation and concurrent changes in the higher structure of DNA. In the nucleus DNA is assembled into chromatin by forming complexes with histone proteins. The structural elements consisting of DNA and histone proteins are called nucleosomes. When a gene is highly transcribed, nucleosomes are fully evicted from the coding region of the gene. Firstly we showed that this eviction is tightly connected to elongating RNAPII and nucleosomes are not removed from the area where RNAPII does not reach. In addition to nucleosomes, RNAPII encounters even bigger obstacles on its way called heterochromatic complexes or silenced chromatin. These complexes have been considered impenetrable for RNAPII. We managed to show that elongating RNAPII is able to remove the heterochromatic complexes and transcribe silenced chromatin. The transcription levels and levels of elongating RNAPII on different genes vary significantly as the requirements for the gene products are not identical. Several genome-wide studies have addressed the question of the distribution of the components of transcriptional machinery. But as these studies draw conclusions on the average signal from the whole cell population they fail to describe processes occurring in a single cell. Thus we aimed to determine the distribution of elongation RNAPII molecules on single chromatin fragment. We found that RNAPII molecules are distributed along the gene evenly. The results obtained from our studies add new information to the basic knowledge of gene transcription and enhance the understanding about the mechanisms of RNAPII-dependent elongation.
In all eukaryotic cells the carriers of genetic information - DNA molecules - are tightly packed to fit in the nucleus. Unpacking DNA is crucial for several cellular processes, including transcription. During RNA polymerase II (RNAPII)-dependent transcription one strand of DNA is used to synthesise complementary mRNA. RNA synthesis is divided into three main phases – initiation, elongation and termination. The main aim of the current thesis was to elucidate the mechanisms of elongation and concurrent changes in the higher structure of DNA. In the nucleus DNA is assembled into chromatin by forming complexes with histone proteins. The structural elements consisting of DNA and histone proteins are called nucleosomes. When a gene is highly transcribed, nucleosomes are fully evicted from the coding region of the gene. Firstly we showed that this eviction is tightly connected to elongating RNAPII and nucleosomes are not removed from the area where RNAPII does not reach. In addition to nucleosomes, RNAPII encounters even bigger obstacles on its way called heterochromatic complexes or silenced chromatin. These complexes have been considered impenetrable for RNAPII. We managed to show that elongating RNAPII is able to remove the heterochromatic complexes and transcribe silenced chromatin. The transcription levels and levels of elongating RNAPII on different genes vary significantly as the requirements for the gene products are not identical. Several genome-wide studies have addressed the question of the distribution of the components of transcriptional machinery. But as these studies draw conclusions on the average signal from the whole cell population they fail to describe processes occurring in a single cell. Thus we aimed to determine the distribution of elongation RNAPII molecules on single chromatin fragment. We found that RNAPII molecules are distributed along the gene evenly. The results obtained from our studies add new information to the basic knowledge of gene transcription and enhance the understanding about the mechanisms of RNAPII-dependent elongation.
Kirjeldus
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Märksõnad
transkriptsioon (biol.), elongatsioon, RNA polümeraas, transcription (biol.), elongation (biol.), RNA polymerase