5S pРНК-связывающие белки бактериальной рибосомы: структура, функция и эволюция тема диссертации и автореферата по ВАК РФ 03.01.03, доктор биологических наук Гонгадзе, Георгий Михайлович

  • Гонгадзе, Георгий Михайлович
  • доктор биологических наукдоктор биологических наук
  • 2010, Пущино
  • Специальность ВАК РФ03.01.03
  • Количество страниц 304
Гонгадзе, Георгий Михайлович. 5S pРНК-связывающие белки бактериальной рибосомы: структура, функция и эволюция: дис. доктор биологических наук: 03.01.03 - Молекулярная биология. Пущино. 2010. 304 с.

Оглавление диссертации доктор биологических наук Гонгадзе, Георгий Михайлович

ВВЕДЕНИЕ.

ГЛАВА I. ОБЗОР ЛИТЕРАТУРЫ.

1. ОТКРЫТИЕ 5S РНК.

2. СТРУКТУРА РИБОСОМНОЙ 5S РНК.

2.1. Первичная структура 5S рРНК и ее эволюция.

2.2. Вторичная структура 5S рРНК или самосборка молекулы.

2.3. Пространственная структура изолированной 5S рРНК.

3. 5S рРНК И РИБОСОМНЫЕ БЕЛКИ.

3.1. Белки, специфически связывающиеся с изолированной 5S рРНК.

3.2. Взаимодействие 5S рРНК и белков в комплексе.

4. 5S рРНК В РИБОСОМЕ.

4.1. Положение 5S рРНК в рибосоме и участие в сборке 50S субчастицы.

4.2. 5S рРНК-белковый комплекс и функционирование рибосомы.

Рекомендованный список диссертаций по специальности «Молекулярная биология», 03.01.03 шифр ВАК

Введение диссертации (часть автореферата) на тему «5S pРНК-связывающие белки бактериальной рибосомы: структура, функция и эволюция»

1. Рибосомные белки Thermus thermophilus, специфически связывающиеся с 5S рРНК.100

1.1. Рибосомные белки TL4, TL5 и TL18 Т. thermophilus образуют прочный комплекс с изолированной 5S рРНК.100

1.2. Белки TL4 (TthL5) и TL18 (TthL18) являются типичными представителями семейств 5S рРНК-связывающихся рибосомных белков L5 и L18, соответственно.104

1.3. Белок TL5 является не только гомологом рибосомного белка L25 Е. coli, но и основного стрессового белка СТС Bacillus subtilis.106

1.4. 5S рРНК-связывающий домен белка TL5.112

2. Формирование 5S рРНК-белкового комплекса Thermus thermophilus

114

2.1. Участок 5S рРНК, с которым взаимодействует белок TL5.114

2.2. Участки 5S рРНК, с которыми взаимодействуют белки TthL5 (TL4) и TthL 18 (TL18).117

3. Пространственная структура бактериального 5S рРНК-белкового комплекса.126

3.1. Подготовка к структурным исследованиям 5S рРНК-связывающих белков Т. thermophilus, а также их комплексов с рРНК.126

3.2. Структура белка TL5 в комплексе со специфическим фрагментом 5S рРНК.129

3.3. Структура белка TthL5 (TL4) в комплексе со специфическим фрагментом 5S рРНК.139

3.4. Структура белка TthL 18 (TL18).148

Заключение.152

4. 5S рРНК-связывающие белки семейства СТС — особенность аппарата трансляции бактерий.154

4.1. Структурная организация белков семейства СТС, их свойства и эволюция.154

4.2. 5S рРНК-узнающий модуль белка семейства СТС.165

5. Участие 5S рРНК-связывающих белков в формировании функционально-активной рибосомы Escherichia coli.172

5.1. Эффект нокаута генов 5S рРНК-связывающих рибосомных белков L5, LI8 или L25 на выживание клеток Е. coli.173

5.2. В отсутствие рибосомного белка L25 клетки Е. coli хотя и выживают, но растут значительно медленнее, чем в его присутствии.178

5.3. Структурные и функциональные свойства рибосом AL25 штамма.181

ЗАКЛЮЧЕНИЕ.192

ВЫВОДЫ.194

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Заключение диссертации по теме «Молекулярная биология», Гонгадзе, Георгий Михайлович

ЗАКЛЮЧЕНИЕ

В завершении этой главы хотелось бы подвести итоги проделанной работы. Нами идентифицированы три рибосомных 5S, рРНК-связывающих белка Т. thermophilus, TL4 (TthL5), TL5 и TL18 (TthL18). Два из них, TthL5 и TthL18, обнаруживают гомологов среди рибосомных белков, всех доменов жизни. Третий белок, TL5 Т. thermophilus, является представителем семейства СТС, которое объединяет только бактериальные белки. Используя результаты, полученные в настоящей работе, а также данные о расшифрованных бактериальных геномах, делается заключение о том, что подавляющее большинство бактерий содержит третий 5S рРНК-связывающий белок, белок семейства СТС. Все белки этого семейства, без исключения, содерж:ат так называемый «5S рРНК-связывающий» домен. Впервые продемонстрировано, что рибосомный белок L25 Е. coli и основной стрессовый белок СТС В. subtilis являются не только структурными, но и функциональными гомологами.

Установлено, что пять идентичных аминокислотных остатков в белках TL5 и L25 формируют их 5S рРНК-узнающий модуль, а учитывая строгую консервативность этих остатков в белках семейства СТС, предполагается, что такой модуль должен быть характерен для большинства из них. Единичные случаи одновременных природных изменений, произошедших в контактирующих областях белков СТС и 5S рРНК, можно считать примерами коэволюции структур этих двух молекул. В данной работе впервые определен сайт связывания рибосомного белка семейства L5 на молекуле 5S рРНК, им оказалась консервативная во всех доменах жизни и уникальная по структуре С-■ петля рибосомной 5S РНК. Сравнительный анализ структур бактериальных и архейных рибосомных белков семейства L5 и их комплексов с РНК позволяет заключить, что структуры этих белков и их контакты с 5S рРНК эволюционно консервативны. Обнаружено, что глобулярная часть пространственных структур рибосомных белков семейств L18 и S11 укладывается практически идентично, что может свидетельствовать об их родственном происхождении. В данной работе определена пространственная структура большей части 5S рРНК-белкового комплекса (более 80%) с высоким разрешением. Эти данные внесли существенный вклад в построение модели рибосомы, и является до сих пор востребованной в исследованиях детальной структурной организации бактериальной рибосомы, так как имеют наилучшее разрешение на сегодняшний день. Получены данные, которые позволили впервые оценить степень необходимости каждого из 5S рРНК-связывающих белков для формирования функционально-активной рибосомы и для выживания бактериальной клетки. Впервые показано, что рибосомные белки L5 и L18 строго необходимы для выживания клеток Е. coli, а белок L25, не являясь ' необходимым для выживания клеток, оказывается чрезвычайно важен для стабилизации структуры центрального протуберанца 50S субчастицы бактериальной рибосомы. Большинство полученных в работе данных имеют приоритетный характер и вносят значительный вклад в понимание тонкой структурной организации бактериальной рибосомы.

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455. Статьи в сборниках и избранные тезисы конференций

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457. Escherichia coli in cell viability and translation // Abstracts of the International Engelgardt conference on molecular biology, august 19-24. 2006. - Buran, Russia. - P.68.

458. Искренне благодарен Станиславу Владимировичу Никонову и сотрудникам его группы, Наталье Невской, Наталье Фоменковой, Роману Федорову и Алексею Никулину, за успешное и приятное сотрудничество в определении структур белков и РНК-белковых комплексов.

459. Благодарю Михаила Бубуненко за приятное и продуктивное сотрудничество. Выражаю большое спасибо Андерсу Лильясу, Торлейфу Харду и Волкеру Эрдману, а также сотрудникам их научных групп, за плодотворное сотрудничество в данной работе.

460. Отдельное спасибо Олегу Никонову за помощь в оформлении работы, а Наталье Леоновой за помощь во всем.

461. Всех сотрудников лаборатории структурных исследований аппарата трансляции благодарю за помощь, участие, ценные советы и просто приятную атмосферу в коллективе.

462. Спасибо сотрудникам Института белка за помощь при выполнении работы. Отдельное спасибо моим родным и близким за величайшее терпение, участие в моих делах, за теплоту и понимание.

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