Structural Biochemistry/Biophysical Studies of Bacterial Ribosome Assembly

Introduction to Bacterial Ribosomes

One of the key processes ribosome carries as a ribonucleoprotein complex is protein synthesis. In the process of ultracentrifugation, the bacterial ribosome sediments as a 70S particle, which is composed of a small subunit of 30S and a large subunit of 50S. The small subunit is composed of a 16S ribosomal RNA and 21 ribosomal proteins, while the large unit is composed of two 23S ribosomal RNA and 33 ribosomal proteins. The small subunit mainly take cares of the association with messenger RNA during the start of translation and decoding, which resolves the 3-nt codon that decides which amino acid to insert to the polypeptide chain. On the other hand, the large subunit acts as the peptidyl transferase center and hence is the site of the formation of peptide bond. The structures of a complete ribosome, including the 30S subunit and the 50S subunit, was solved to confirm that the ribosome is actually a ribozyme and the fact that catalytic sites indeed lie within ribosomal RNA components. These structures are essential factors in determining the mechanism of translation, but information about how the ribosome assembles into a stable multi-component complex is still very limited. Ribosome assembly is the core of ribosome genesis, which involves the following steps:

(1) the appropriate folding or ribosomal ribosomal RNA and ribosomal proteins

(2) the binding and unbinding of assembly factors

(3) the modification and translation of ribosomal proteins

(4) the binding of ribosomal proteins

(5) the processing, modification, and transcription of ribosomal RNA

Ribosomal assembly mechanisms

Most of these steps are carried simultaneously with the transcription of the ribosomal RNAs, and the ribosome assembly process is carried through protein-binding events and RNA conformational changes. At these events, the binding of ribosomal proteins stabilizes the RNA folding process and eventually drives the RNA structure to the final native state. Overall, ribosome is an important protein structure used to study principles of RNA, RNA protein recognition, protein folding, and the assembly of multi-component complexes. Incorrectly assembled ribosomes may give rise to diseases, therefore studying ribosome biogenesis may offer insights in developing more efficient ways to treat diseases. It is essential for scientists to understand both temporal and physical pathways of intact ribosomes because that is how they can gain knowledge regarding the regulation and possible errors happened during the assembly of bacterial ribosome.

General Information

The structure of bacterial ribosomes is composed of over 50 proteins and three large domain RNA molecules. Modifications in the rRNA require dozens of gene products but the role of these modifications in ribosome function are not fully understood or seem nonessential. It is believed that these modifications are a part of stabilizing RNA structure or RNA-protein interactions, mediate translation, or as checkpoints in ribosome assembly. The development of certain biophysical methods have helped better the understanding of how bacterial ribosome was constructed along with how its structure leads to function. Ribosome assembles improperly can lead to various diseases in human body because ribosomes assembly plays an important role in cells as RNA protein recognition. Therefore, understanding the ribosome assembly is a need to see how they connect together. Studies how ribosomes are regulated helps to figure out how and why errors occur in assembly biogenesis. The three contributions have come from mass spectrometry, computational methods, and RNA folding studies. The metabolism of the bacterial ribosomes helps ensure the proper and timely synthesis of other components. Because ribosome is the large and complex macromolecule, proper product is important thus require an efficient steps during assembly process. The table below represents an order of events that happen during assembly of the ribosome but not necessarily in that exact order. All those events must occur but the pathway can be somewhat random and vary throughout nature.

The assembly of bacterial ribosomes is thought to progress through an alternating series of RNA conformational changes as well as protein-binding events. The assembly of these bacterial ribosomes occurs much faster and efficiently in vivo than in vitro.

1) Transcription of Ribosomal RNA- rRNA are transcribed as single transcription. 2) Synthesis of ribosomal proteins-about 55 ribosome protein synthesized in both transcriptional and post-transcriptional gene regulation mechanisms. 3) Ribosomal RNA modification-Ribosome proteins are modified at about 30-40 specific positions either by base methylation and/or pseudouridylation. The role of the assembly is to stabilize RNA structure or RNA protein interactions. 4) Ribosomal protein modification. 5) Ribosomal RNA processing-produce mature rRNA 6) Ribosomal RNA folding cotranscriptionally 7) Ribosomal protein binding cotranscriptionally 8) Assembly factor binding and release- bind at various times that promotes the orderly and efficient process of assembly.

In Vitro Assembly Map and Assembly Intermediates

With advancing technologies that can easily determine the structure of ribosome with high resolution, experimental researches that were done in the previous decades were somewhat neglected. Instead of focusing on the structure of ribosome, these studies focused on determining the components of the ribosome. In the late 1950s, researchers came to the conclusion that various subunit components of ribosomes were actually different physical states of the same particle, which were regulated by the concentration of magnesium ions. Tissieres and Watson successfully demonstrated that the bacterial ribosome is composed of a 70S particle that could be either aggregated to form the 100S particle or broken down to the large and small subunits. Later in the 1960s, Traub and Nomura took a huge step forward. They demonstrated that additional components are required for an active 30S subunit to be assembled in vitro from free ribosomal RNA and ribosomal proteins. In another words, the ribosomal RNA and ribosomal proteins possess all of the information needed for the assembly of ribosomes. Recombinant ribosomal proteins, unmodified 16S ribosomal RNA transcribed in vitro, and 16S ribosomal RNA in combination with purified proteins can reconstitue to 30S particles. Also, the 30S particle contains three domains, including the platform region, the head, and the body; each domain of this subunit can be independently reconstituted. This piece of information is very significant because it proposes the possibility to see through the process of ribosome assembly simply by changing various components. Although this simple perspective from earlier researches seemed questionable to some scientists, it was a solid foundation for ongoing researches on ribosome assembly and biogenesis.

Mass Spectrometry

The three main applications of using mass spectrometry with ribosome are inventory of the different components, identifying modifications of ribosomes, and study of ribosome dynamics. The ability to map modifications has allowed scientists to study the changes within modifications as the species develops. This can pinpoint the specific role protein modifications have in assembly or translation. The analysis of isolated ribosomes is the only way to completely characterize modifications for new species. Ribosomal modifications leads to a specific genotype and studying the genotype can give hints to what the role of that ribosomal protein modification has in assembly or translation. iTRAQ mass spectrometry technique is used to examine the mutation in the assembly cofactor. The molecular weigh and the sequence of ribosome protein can be determined using this Mass spectrometry.The HDX application reveals which region of bound ribosomal protein is the most flexible because these regions are important during the translation step.

Computational Methods

The large number of atoms in ribosomes makes this method a challenge. Through this way, the flexibility of protein binding sites has been shown to directly correlate with the stage at which a protein binds there. Primary structure proteins bind at sites that are more rigid while secondary and tertiary structures bind on sites that are more flexible. In addition, computational methods has shown that the order of assembly is guided by electrostatic binding. These energies directly reflect changes in flexibility and electrostatics.

RNA folding and Ribosomal Binding, in vitro

Biochemical method provides dept understanding of the series of conformational changes that occur during the assembly process. The fastest folding regions is said to happen at the primary protein-binding sites and this binding site is required to produce mature ribosome protein subunits. The role of the fast self-folding of the 5’ domain is to be synthesized cotranscriptionally in assembly process. It is possible to assemble and examine the conformation change of ribonucleoprotein using reconstitution intermediate (RI) with a subset of 15 ribosomal proteins in which reconstitution reactions were carried out at low temperatures. The conformational change of RI intermediate occurred by heating up. This then form a new structure (RI*) that has the ability to bind the remaining five proteins and form 30S subunits. The flexibility of protein binding reaction is due to the differences in the accessibility of the RNA to probes at different temperatures. Also, protein is binding at different regions of the rRNA at different times due to the intervening RNA folding reactions. The temperature of the binding rates is dependent. This indeed revealed a unique kinetic barrier to binding of each ribosome protein.

References

James R. Williamson, Biophysical Studies of Bacterial Ribosome Assembly, Curr Opin Struct Biol. 2008 June ; 18(3): 299–304.

James R. Williamson, Zahra Shajani, Michael T. Sykes. Assembly of Bacterial Ribosomes. Annual Review of Biochemistry. 26 April 2011. 80: 501-26.