Ribosomes are molecular machines that make proteins in cells. That the ribosomes are important can be judged by the fact that the cells spend about 40% of their energy in assembling them. In bacteria, ribosomes are made up of a large (50S) and a small (30S) subunits. Flaws in the assembly and maturation (biogenesis) of any of these subunits affect protein synthesis in various ways and often result in the organism’s intolerance to cold, and impact their resistance to drugs and pathogenity. In higher organisms (including humans), defective biogenesis of ribosomes could lead to various diseases. Hence, an understanding of how cells manage accuracy in the complex process of ribosome biogenesis is of utmost importance in developing therapeutic interventions. Now, a study from the laboratory of Prof. Umesh Varshney at the Department of Microbiology and Cell Biology, Indian Institute of Science (IISc), Bangalore, has unravelled the mechanism behind synthesis of ribosomes.
“Ribosomopathies are disorders that occur due to defective ribosome biogenesis/maturation. Our study has discovered a new step in the ribosome maturation pathway. These studies provide us with a good model to understand how the ribosomes that do not mature, affect translation of various mRNAs in the cell”, explains Prof. Varshney, describing the application of this research. Using E. coli, a type of bacteria, the researchers have studied the role of a type of RNA known as initiator transfer RNA (i-tRNA) in the formation of ribosomes and the effects of immature ribosomes on protein synthesis.
Expression of the genetic information in organisms occurs mainly in two stages. In the first stage, called transcription, the information contained in the cell’s DNA is transferred to a “messenger” RNA (mRNA). Protein synthesis actually occurs in the second stage called translation, which is sub-divided into three steps - initiation, elongation, and termination. At the initiation step, the small subunit of the ribosome binds with the i-tRNA and the mRNA at its “start” location, and joins with the large subunit of the ribosome with the help of many small proteins. This collaborative set up of the ribosomal subunits, mRNA and i-tRNA ensures correct encoding of the genetic information in the mRNA into protein. In the elongation step, the ribosome makes the protein by bringing together the building blocks of protein - amino acids- in response to the information available in the mRNA. The termination step is reached when the ribosome encounters the “stop” location on the mRNA, thus bringing the process to a stop. This step is then followed by a process that separates the two subunits of ribosomes so as to make them available for another round of protein synthesis.
In the current investigation, the researchers carried out an indepth study using Northern blotting, a technique used to detect the RNAs of interest from the entire RNA mix, on two strains of E. coli - a wild type strain and a mutant strain with three of its four i-tRNA genes removed. The mutant strain showed sensitivity to cold and the researchers also observed an increased accumulation of immature 16S rRNA which is a sub-component of the 30S (the smaller subunit) ribosome. Further, when the missing i-tRNAs were genetically introduced into the mutant strain, both cold insensitivity and the maturation flaws in 16S rRNA were corrected.
The researchers used specific inhibitors to halt translation in cells at different stages. They found that most of the inhibitors did not significantly impede 16S rRNA maturation; however, the inhibitors that interfered with i-tRNA binding to the 30S ribosome did, highlighting the importance of i-tRNA dependent ribosome maturation during the pioneering round (the first time a newly assembled 30S ribosome is used) of translation initiation. “Pioneering round of initiation seems to flag the culmination of the ribosome maturation process”, suggests Dr. Varshney.
This study provides a model to study the role of i-tRNA in ribosome biogenesis and also alludes to the translation stage at which maturation occurs. “We will now follow up how deficiency of i-tRNA affects translation of the mRNA pool in the cell. Deficiencies in ribosome maturation would result in the generation of heterogenous pool of ribosomes which could translate the mRNA pools differentially and result in an altered proteome in the cell”, concludes Dr. Varshney, talking about the future course of research.