Роль элементов 23S pРНК в формировании сайтов связывания тРНК на рибосоме Escherichia coli и в процессе транслокации тема диссертации и автореферата по ВАК РФ 02.00.10, кандидат химических наук Лесняк, Дмитрий Витальевич

• Лесняк, Дмитрий Витальевич
• кандидат химических наук
• 2007

Оглавление диссертации кандидат химических наук Лесняк, Дмитрий Витальевич

СОДЕРЖАНИЕ.

СПИСОК СОКРАЩЕНИЙ.

ВВЕДЕНИЕ.

1. ОБЗОР ЛИТЕРАТУРЫ.

1.1. Структура 70S частиц Escherichia сои.

1.2. Связывание тРНК с рибосомой.

1.3. Расположение молекул тРНК в различных функциональных состояниях рибосомы.

1.3.1. А/Т положение тРНК.

1.3.2. А/А положение тРНК.

1.3.3. Р/Р положение тРНК па рибосоме.

1.3.4. Е/Е-положение тРНК на рибосоме.

1.4. Изменение конформации тРНК во время элонгационного цикла.

1.5. Гибридные сайты связывания тРНК.

1.6. Исследования динамики тРНК на рибосомах методом флуоресцентного резонансного переноса энергии.

1.7. Структура рибосомы при гибридном положении тРНК.

1.8. Эволюция представлений о структуре и функции Е-сайта.

1.9. Обратная транслокация тРНК.

Введение диссертации (часть автореферата) на тему «Роль элементов 23S pРНК в формировании сайтов связывания тРНК на рибосоме Escherichia coli и в процессе транслокации»

Рибосома - сложная молекулярная машина рибонуклеопротеидной природы. В клетках всех живых существ она отвечает за синтез белка. Процесс биосинтеза белка занимает центральное место в молекулярной биологии, и история его изучения насчитывает уже более половины столетия. За это время значительно возросли возможности исследователей, умножились знания во всех областях науки, ускорились средства получения и обработки экспериментальных данных, но количество работ, посвященных изучению работы рибосомы, только возрастает. В рибосомологии осталось много загадок.

Одной из наиболее интригующих тайн является транслокация мРНК и тРНК на рибосоме. Это активный, динамичный процесс, для понимания которого мало знать атомарную структуру рибосомы и ее комплексов с мРНК и тРНК. Необходимы глубокие структурно-функциональные исследования, воспроизведение промежуточных стадий процесса in vitro и их изучение, выяснение роли отдельных элементов всего молекулярного ансамбля в транслокации.

В настоящей работе мы изучали свойства Е-сайта рибосомы с помощью сайт-направленного мутагенеза, а также выясняли роль малоизученного участка пептидилтрансферазного центра в цикле трансляции. Нами были сделаны две точечные замены в 23 S рибосомной РНК, которые оказали серьезное влияние на жизнеспособность мутантных штаммов Escherichia coli. Мутации были нацелены на заранее определенные этапы процесса транслокации. Одна из введенных мутаций привела к сильному снижению жизнеспособности клеток, вторая оказалась летальной. Работа посвящена изучению отдельных стадий функционирования рибосомы, которые оказались нарушены в результате данного мутагенеза. Основные эксперименты сделаны in vitro на очищенных компонентах аппарата трансляции. Обзор литературы посвящен положениям тРНК на рибосоме на различных этапах элонгационного цикла и рассмотрению современных моделей транслокации мРНК и тРНК на рибосоме.

1. Обзор литературы

Заключение диссертации по теме «Биоорганическая химия», Лесняк, Дмитрий Витальевич

4. Выводы

1. Мутация С2394в 23Б рРНК нарушает связывание деацилированной тРНК с Е- и Р/Е-сайтами рибосомы.

2. Отсутствие стабильного связывания тРНК с Е- и Р/Е-сайтами рибосомы приводит к увеличению частоты сдвига рамки считывания и прочтения стоп-кодонов как смысловых.

3. Связывание тРНК с гибридным Р/Е-сайтом рибосомы необходимо для стимуляции СТРазной активности ЕР-в.

4. Мутация в2061С 23Б рРНК летальна. Она приводит к изменению константы связывания РЬе-тРНКРЬс с рибосомой, ингибирует пептидилтрансферазную реакцию с пуромицином и приводит к изменению конформации пептидилтрансферазного центра.

5. Нуклеотид в2061 23Б рРНК необходим для функциональной активности белка ЯМЕ.

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