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This volume provides comprehensive reviews and describes the latest techniques to study eukaryotic ribosome biogenesis. For more than 50 years ribosomes are a major research topic. Our knowledge about ribosome biogenesis and function such as transcription, mRNA modification, and translation was the sine qua non for developing the powerful RNA-based vaccines against RNA-viruses causing the world-threatening Covid-19 pandemia. The chapters in this book are organized into six parts. Part One discusses a comparative survey about the unity and diversity of ribosome biogenesis in pro- and eukaryotic cells. Part Two deals with the genomic organization of eukaryotic rDNA and the role of RNA polymerase I in ribosomal RNA transcription. Part Three explores in vitro methods to study RNA polymerase I structure and its function, and Part Four analyzes the nucleo-cytoplasmic transport of assembled ribosomes and RNP complexes. Part Five covers modifications that increase the complexity of rRNAs, and Part Six provides readers with a review of eukaryotic translation and - for the first time - describes a new method to analyze translation in vitro. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Ribosome Biogenesis
A Comparative Perspective on Ribosome Biogenesis:​ Unity and Diversity Across the Tree of Life
Genomic Organization
Establishment and Maintenance of Open Ribosomal RNA Gene Chromatin States in Eukaryotes
Analysis of Yeast RNAP I Transcription of Nucleosomal Templates In Vitro
RNA Polymerases
Specialization of RNA Polymerase I in Comparison to Other Nuclear RNA Polymerases of Saccharomyces cerevisiae
Structural Studies of Eukaryotic RNA Polymerase I Using Cryo-Electron Microscopy
Preparation of RNA Polymerase Complexes for Their Analysis by Single-Particle Cryo-Electron Microscopy
Ribosome Assembly, Transport and RNP Complexes
Eukaryotic Ribosome assembly and Nucleocytoplasmi​c Transport
Tethered MNase Structure Probing as Versatile Technique for Analyzing RNPs Using Tagging Cassettes for Homologous Recombination in Saccharomyces cerevisiae
RNA Modification
Chemical Modifications of Ribosomal RNA
In Vitro Selection of Deoxyribozymes for the Detection of RNA Modifications
Mapping of the Chemical Modifications of rRNAs
Non-radioactive In Vivo Labeling of RNA with 4-Thiouracil
Translation
Translation Phases in Eukaryotes
Differential Translation Activity Analysis Using Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) in Archaea
Thermofluor-Based Analysis of Protein Integrity and Ligand Interactions
In Vitro Assembly of a Fully Reconstituted Yeast Translation System for Studies of Initiation and Elongation Phases of Protein Synthesis