Взаимодействие тмРНК с рибосомой в процессе транс-трансляции тема диссертации и автореферата по ВАК РФ 02.00.10, кандидат химических наук Бугаева, Елизавета Юрьевна

• Бугаева, Елизавета Юрьевна
• кандидат химических наук
• 2008

Оглавление диссертации кандидат химических наук Бугаева, Елизавета Юрьевна

Содержание

Список сокращений

Введение

Обзор литературы тмРНК

Общие сведения о тмРНК

Вторичная структура тмРНК

Процессинг тмРНК

Модификация оснований тмРНК

Аминоацилирование тмРНК

Белок SmpB

Общие сведения о SmpB

Пространственная организация SmpB

Взаимодействие белка SmpB с тмРНК

Взаимодействие SmpB и тмРНК с рибосомой

Транс-трансляция

Модель процесса тиранс-трансляции

Причины появления свободного А сайта в транслирующих рибосомах

Распознавание «арестованных» рибосом и свободного А сайта 43 Переключение на матричную область, узнавание кодона продолжения синтеза

Элонгация и терминация

Деградация белка и мРНК

Биологическая роль /ядонс-трансляции

Материалы и методы

Реактивы и биопрепараты

Буферы и растворы

Трансформация клеток Escherichia coli 57 Приготовление компетентных клеток Escherichia coli для трансформации с помощью теплового шока

Трансформация с помощью теплового шока 58 Введение мутаций в ген, кодирующий тмРНК. Сайт-направленный мутагенез

Анализ активности мутантных молекул тмРНК с помощью „генетической системы"

Выделение комплекса тмРНК с рибосомой

Выделение суммарной РНК комплекса

Окрашивание ПААГ

Выделение суммарного белка комплекса

Окрашивание ПААГ-SDS Кумасси R

Масс-спектрометрический анализ белков

Диализ

УФ-сшивание тмРНК с рибосомой

Приготовление зонда для гибридизации с тмРНК

Нозерн-блот анализ

Переосаждение антител

Определение концентрации белка по методу Брэдфорда

Вестерн-блот анализ

Выделение мутантных тмРНК

Химический пробинг

Крио-электронная томография

Компьютерное моделирование

Результаты и обсуждение

Выделение комплексов транспортно-матричной РНК с рибосомой

Анализ РНК, входящих в состав выделенных комплексов

Анализ белкового состава выделенных комплексов 79 Исследование комплекса тмРНК с рибосомой методом ковалентного сшивания 83 Исследование комплексов тмРНК с рибосомой методом химического пробинга 87 Исследование комплекса тмРНК с рибосомой методом крио-электронной томографии

Компьютерное моделирование процесса прохождения тмРНК через рибосому в ходе транс-трансляции

Выводы

Заключение диссертации по теме «Биоорганическая химия», Бугаева, Елизавета Юрьевна

выводы

1. Во всех выделенных тмРНК с рибосомой, в которых транс-трансляция остановлена на различных этапах, присутствует белок SmpB. Стехиометрия комплексов рибосома-тмРНК-SmpB равна 1:1:1 на всех стадиях транс-трансляции. Белок SmpB занимает канонический сайт связывания с тРНК-подобным доменом тмРНК.

2. Псевдоузел 3 тмРНК находится на поверхности рибосомы на всех этапах транстрансляции.

3. Псевдоузлы 2, 3 и 4 образуют петлю на поверхности 70S рибосомы. Эта петля подвижна в растворе и может занимать различные положения от «плеча» до вершины «головы» 30S субчастицы рибосомы. Положения концов данной петли тмРНК на рибосоме соответствуют входу и выходу канала мРНК, предполагая размещение в нем мРИК-подобной части тмРНК.

4. Структура и контакты тмРНК, за исключением мРНК-подобного домена и спирали 5, практически не изменяются в ходе транс-трансляции. Спираль 5 стабилизируется или защищается при взаимодействии тмРНК с рибосомой. Спектр модификаций нуклеотидов мРНК-подобного домена изменяется по мере прохождения тмРНК через рибосому в ходе транс-трансляции.

5. Изменения последовательности в мРНК-подобном домене тмРНК влияют на реакционную способность нуклеотидов в спирали 2, что предполагает существование третичного контакта между этими областями.

6. Полученные данные позволили предложить механизм прохождения тмРНК через рибосому в ходе транс-трансляции.

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