Функциональная топография транспортно-матричной РНК тема диссертации и автореферата по ВАК РФ 02.00.10, доктор химических наук Шпанченко, Ольга Валерьевна

• Шпанченко, Ольга Валерьевна
• доктор химических наук
• 2010

Оглавление диссертации доктор химических наук Шпанченко, Ольга Валерьевна

СПИСОК СОКРАЩЕНИЙ.

ВВЕДЕНИЕ.

ОБЗОР ЛИТЕРАТУРЫ.

Транспортно-матричная РНК.

Вторичная структура тмРНК.

Процессинг предшественника тмРНК.

Модифицированные основания в тмРНК.

Аминоацилирование тмРНК.

Белок БтрВ.

Пространственная организация белка БтрВ.

Структура комплекса тРНК-подобной области тмРНК с белком ЭтрБ.

Структурная организация комплексов тмРНК и белка ЭтрВ с рибосомой.

Транс- трансляция.

Аминоацилирование тмРНК, её взаимодействие с белками БтрВ и ЕБ-Ти, а также связывание этого комплекса с рибосомой.

Причины появления свободного А-участка в транслирующих рибосомах.

Распознавание «арестованных» рибосом и свободного А-сайта.

Переключение на матричную область, узнавание кодона продолжения синтеза.

Элонгация и терминация.

Деградация белка и мРНК.

Биологическая роль /и/7ш/с-трансляции.

РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ.

Неканоническое взаимодействие тмРНК с элонгационным фактором Ти.

Синтез модифицированной тмРНК.

Формирование и анализ ковалентного соединения тмРНК с белками Б100 экстракта.

Комплекс деацилированной тмРНК с элонгационным фактором Ти в присутствии вТР или вБР.

Модель взаимодействия тмРНК с рибосомой.

Двухдоменная организация тмРНК.

Поиск элемента тмРНК, обеспечивающего узнавание А-участком рибосомы

Исследование роли некоторых консервативных нуклеотидов тмРНК.

Поиск интрамолекулярных супрессоров для летальных мутаций ЦА85,86СС иЦАЗ()0,301СС.

Мутации нуклеотидов тРНК-подобной части, затрагивающие активность тмРНК.

Выделение комплексов транспортно-матричной РНК с рибосомой.

Анализ РНК, входящих в состав выделенных комплексов.

Анализ белкового состава выделенных комплексов.

Исследование комплексов тмРНК с рибосомой методом химического пробинга.

Исследование комплекса тмРНК-4 с рибосомой методом криоэлектронной томографии.

Моделирование структуры комплексов тмРНК с рибосомой.

МАТЕРИАЛЫ И МЕТОДЫ.

Реактивы и биопрепараты.

Буферы и растворы.

Олигодезоксирибонуклеотиды и штаммы.

Фотоаффинное химическое сшивание тмРНК.

Синтез тмРНК с помощью Т7-РНК-полимеразы.

Синтез тмРНК, содержащей 4-тиоуридин, с помощью Т7-РНК-полимеразы

То-фингерпринты.

Комплексообразование и фотоактивируемое химическое сшивание тмРНК

Анализ белкового компонента «сшивки».

Определение позиции «сшивки» в тмРНК.

Футпринтинг.

Внутримолекулярное фотоаффинное^химическое сшивание тмРНК.

Анализ продуктов сшивания с помощью реакции обратной транскрипции

Гидролиз тмРНК с помощью РНКазы Н.

Измерение констант ассоциации комплексов тмРНК и тРНК с EF-Tu.

Манипуляции с ДНК.

Приготовление компетентных клеток Е. coli для трансформации с помощью теплового шока.

Трансформация компетентных клеток Е. coli с помощью теплового шока 163 Приготовление компетентных клеток Е. coli для электротрансформации

Электротрансформация клеток Е. coli.

Выделение плазмидной ДНК.164

Определение концентрации ДНК в растворе.

Разделение фрагментов ДНК в агарозном геле.

Выделение фрагментов ДНК из агарозного геля.

Приготовление векторов и вставок.

Лигирование.

Сайт-направленный мутагенез.

Создание штамма Е. coli SKZl(Assr./4).

Клонирование гена, кодирующего тмРНК.

Создание плазмиды pR для генетической системы определения активности тмРНК.

Конструирование плазмиды pGEMstra.

Сайт-направленный мутагенез тмРНК.

Клонирование гена репортёрного пептида в плазмиды семейства pGEM.

Случайный мутагенез тмРНК in vivo и анализ мутантов.

Определение первичной структуры ДНК по Сэнгеру.

Анализ активности молекул тмРНК.

Анализ активности молекул тмРНК с помощью «генетической» системы. 173 Определение активности молекул тмРНК с помощью гибридного фага

XimmP22.

Аминоацилирование тмРНК.

Анализ положения пептида в комплексе.

Выделение и анализ комплексов тмРНК с рибосомой.

Выделение комплексов.

Анализ РНК-состава комплекса.

Анализ белкового состава комплекса.

Изучение комплексов тмРНК с рибосомой методом химического пробинга.184 Определение структуры комплекса рибосомы с тмРНК-4 методом криоэлектронной томографии.

Моделирование структуры комплексов тмРНК с рибосомой.

ВЫВОДЫ.

Заключение диссертации по теме «Биоорганическая химия», Шпанченко, Ольга Валерьевна

выводы

1. Элонгационный фактор Tu и тмРНК участвуют во взаимодействии нового типа. В отличие от канонического комплекса между ацилированным тРНК-подобным доменом тмРНК и EF-Tu*GTP деацилированная тмРНК образует комплекс с EF-Tu*GDP за счёт взаимодействия со спиралью 2 и псевдоузлом 4.

2. Белок SrapB входит в состав тмРНК-рибосомных комплексов на всех стадиях транс-трансляции. Элонгационные комплексы содержат по одной молекуле белка SmpB, связанной с каноническим участком взаимодействия на TLD тмРНК.

3. Молекула тмРНК в растворе существует в виде двух компактно свернутых доменов, между которыми уложена одноцепочечная мРНК-подобная область. В ходе транс-трансляции домены, не изменяя своей структуры, занимают определённые сайты на рибосоме, в то время как матричная область перемещается по ней в экспонированной форме.

4. Экспериментальная проверка двухдоменной модели с помощью химических, физических и биоинформационных подходов позволяет построить динамическую модель транс-трансляции, адекватную всей совокупности имеющихся на' сегодняшний день данных. Согласно этой модели структурные элементы тмРНК (псевдоузлы, спирали, арка вокруг головы 30S рибосомной субчастицы, сформированная псевдоузлами), сохраняются в процессе шранотрансляции. Основные изменения при прохождении MLD тмРНК через рибосому затрагивают район мРНК-подобной области и спирали 5 тмРНК.

5. Правильное позиционирование ко дона возобновления синтеза в А-участке рибосомы задается структурным элементом, сформированным белком SmpB, псевдоузлом 1 и петлёй А79-А86 тмРНК, который совершает поворот при транслокации TLD тмРНК из А-участка рибосомы в Р-участок.

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