Исследование бинарных комплексов РНК и рибосомного белка S7 эубактерий тема автореферата и диссертации по химии, 02.00.10 ВАК РФ

2 ВВЕДЕНИЕ.

3 ОБЗОР ЛИТЕРАТУРЫ. РИБОСОМНЫЙ БЕЛОК S7 ЭУБАКТЕРИЙ,КТУРА И ФУНКЦИЯ.

3.1 Первичная структура рибосомного белка S7 эубактерий.

3.2 Высшие структуры белка S7 эубактерий.

3.3 Белок S7 - компонент малой субчастицы рибосомы.

3.4 Белок S7 - репрессор сопряженной трансляции str оперона.

4 РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ.

4.1 Компьютерное моделирование третичной структуры EcoS7 и его комплексов с 16S рРНК.

4.2 Конструирование супер-продуцента Е. coli для белка EcoS7 и выделение EcoS7 и TthS7.

4.3 Клонирование EcoStr мРНК и получение фрагментов РНК.

4.4 Взаимодействие фрагмента Eco16S рРНК с белками EcoS7 и

TthS7.

4.5 Идентификация мест контактов белков EcoS7 и TthS7 с фрагментом Eco16S рРНК методом УФ-индуцируемой «сшивки» с нулевой длиной.

4.6 Изучение влияния белков EcoS7 и TthS7 на структуру фрагмента Eco16S рРНК.

4.7 Взаимодействие фрагмента EcoStr мРНК с белками EcoS7 и

TthS7.

4.8 Идентификация мест контактов белков EcoS7 и TthS7 с фрагментом EcoStr мРНК методом УФ-индуцируемой «сшивки» с нулевой длиной.

4.9 Изучение влияния белков EcoS7 и TthS7 на структуру фрагмента EcoStr мРНК.

5 ЭКСПЕРИМЕНТАЛЬНАЯ ЧАСТЬ.

5.1 Используемые штаммы микроорганизмов и реактивы.

5.2 Используемые методы.

6 ВЫВОДЫ:.

6 ВЫВОДЫ:

1. Методами компьютерного анализа и моделирования in silico показано, что два белка-аналога EcoS7 и TthS7 по первичной структуре идентичны на 52%, а в зоне РНК-белковых контактов в 30S субчастице - на 83%. Подобная высокая консервативность дает возможность экспериментально сравнить свойства гомологичных и гетерологичных РНК-белковых комплексов, причем TthS7 можно рассматривать как «природный мутант» EcoS7.

2. Получен супер-продуцент Е. coli для EcoS7. Показано, что для стабильной экспрессии регуляторного белка необходимы системы с самым жестким контролем регуляции экспрессии (pET28b/BL21 DE3 pLysE).

3. Установлено, что взаимодействие in vitro EcoS7 и TthS7 с фрагментом 16S рРНК, в целом однотипно, но отлично от таковых в 30S субчастице рибосом.

4. Установлено, что белок S7 взаимодействует с участком бифуркации межцистронного фрагмента S12-S7 str мРНК, что приводит к изменению конформации в основании шпильки, где расположены сигналы трансляции. На основании этого предложена гипотеза о механизме регуляции сопряженной трансляции.

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