Researchers from Hopkins and the University of Maryland have uncovered the source behind the amazing stability of RNA transcripts that regulate the expression of genes in cells.
Ribonucleic acid, or RNA, is one of the three macromolecules essential for the expression of genes. It is the intermediate step in the Central Dogma of Biology, which describes how DNA genes are transcribed to coding RNA, or messenger RNA, which is then translated into proteins, whose activity in cells facilitates their basic functions.
Broadly, RNA can be divided into two categories: coding RNA and non-coding RNA. Non-coding RNA has many regulatory functions, each related to the unique 3D structures it can form. RNA exists as one strand, unlike the famous double helix of DNA, which is composed of two strands. The single-stranded RNA can create bonds with itself at different locations to create numerous 3D structures.
These structures help it regulate gene expression and catalyze biochemical reactions that are associated with proteins. The most important RNA-protein association is the ribosome, which is responsible for catalyzing the process by which coding RNA is used to build protein structures.
By mutating these ribosomes, researchers were able to investigate the relationship between 3D RNA structures and the sequences in their structure that allow their formation. They chose a ribosome whose sequence has already been characterized, had a stable structure and could tolerate a relatively high degree of mutation in its sequence.
The most common structures formed in RNA are double helices, similar to the structure of DNA first described by James Watson and Francis Crick. These are held together by tertiary interaction motifs, which are sequences of nucleotides in the RNA itself. How these helices are oriented within the 3D RNA structure is important for its function. The tertiary interaction motifs are therefore extremely important in RNA stability.
They created a series of mutants that were all functional to different degrees; some were even 200 percent more active than the original. Despite this, the mutated RNA transcripts had sequences that made them more energetically favorable for them to remain unfolded.
They then allowed the mutated RNAs to fold, while exposing them to magnesium ions. Using the general rule of RNA folding - which states that the more stable the structure, the less magnesium is required for its formation - the scientists were able to calculate the stability of the resulting 3D structures.
They found that single mutations destabilized both the complete, or native, folded form of the RNA, as well as an intermediate folding step. More importantly, double mutations showed that there is a framework by which the tertiary interaction motifs cooperate with each other to increase the stability of the folded RNA beyond what it would be if they acted independently of each other.
This was proved by the change in energy state between the folded and unfolded forms, as characterized by the number of magnesium ions the RNA used to maintain its folded state. The energy coupling of the tertiary motifs is larger than the energy of the individual tertiary structures.
This cooperation only occurred during the folding process and was not present in the final RNA construct; therefore it only affected the intermediate structure of the mutants.
The researchers postulate that this cooperation is a result of natural selection of RNA structures that were conducive to forming a stable folded structure. This was supported by their observation that random sequences in RNA did not have the folding capabilities of more evolved sequences, even when they had similar composition of nucleotides.