Professor Ming Hammond
08-24-2010
Project Title: Gene Control by Small Molecules Binding to Modified mRNA Transcripts.
It is extremely difficult to put thoughts down onto paper all the time, but I will do it anyway. I was embarrassed of my short and muddled oral dictation to Ming Hammond of ideas sparked by tree sap. I was inspired to test the flammability of tree sap yesterday while running along the easy bay lagoon park. I stopped to observe the sticky droplets of liquid forming on the bark of a conifer and realised how good the oil smelled and how energy dense the oil must be since it consists of extremely long chains of unsaturated hydrocarbons. I am struck by the large amount of unique plant liquids, gels, and solids present in such a small local area close to my home. I feel that all of the solutions to our world's problems lies in the things which are in close proximity.
'Wiki'ing things like Ribosome, DNA, and Genes would likely be a good substitute to reading this next part or you can make your way through my explanations.
Today I had a meeting with Professor Hammond and her group members where I was told about a graduate student's project involving the use of messenger ribonucleic acid (mRNA) to control gene expression in plants via differential gene splicing. Keep in mind that mRNA is transcribed from helical double-stranded deoxyribonucleic acid (DNA) to make a single-stranded replica of DNA. This single-stranded replica is made up of the same three nucleotide bases as DNA, Adenine (A), Guanine (G), and Cytosine (C), except for that RNA replaces the fourth DNA nucleotide base Thymine (T) with Uracil (U). Uracil (U) differs from Thymine by the absence of only one methyl (-CH3) group which to an Organic Chemist is not much. There is one other thing that RNA has that DNA does not which gives it the different name and that is the presence of a hydroxyl (OH) group on the ribose sugar ring to which the individual nucleotides are connected. Thus, DNA is called deoxyribonucleic acid. Cells use single stranded RNA 'transcripts' of DNA as a translation template in order to link amino acids together and synthesize proteins and enzymes. Ironically, RNA is translated into protein using protein-RNA complexes (ribonucleoproteins) called ribosomes.***
A gene is chunk of DNA which encodes transcriptional information for the synthesis of mRNA and thus by creating mRNA ultimately provides the translational information for the synthesis of proteins. Proteins are what give cells their amazing abilities to transform their environment into whatever they desire.
Going back to the basis of the project which Ming discussed. One can insert particular RNA sequences smack dab in the middle of a protein-coding gene and control the expression or production of the protein that that particular gene encodes. This means you can turn the gene on and off like a switch and produce, for example, a plant that degrades its own cellulose on command with the addition of a chemical signal.
The RNA sequence inserted binds a specific protein causing changes in the post-transcriptional splicing, a term which refers to how the mRNA code is trimmed and ordered before translation in order to produce a working protein. The cell uses the additional step of RNA splicing to regulate its own production of proteins, this is a recurring theme in biology and regulation is provided in many other ways including transcription factors, small molecule inducers, and DNA binding proteins*.
In this project, RNA sequences that bind specifically to one type of protein are inserted between particular amino acid sequences of the protein-coding gene, for example between the codons that encode for the two residues Glutamate (E) and Arginine (R) in a sequence. The protein-binding RNA can bind specifically to a protein called L5. When L5 protein is not bound to the RNA binding site a premature stop codon is spliced into the final mRNA transcript. A stop codon is a sequence of mRNA that tells the ribosome to stop building protein and to dismount from the mRNA strand.
The premature stop codon is located in the engineered protein-binding RNA sequence and tells the ribosome to stop translating earlier than is usual if L5 activator is not present. Thus, the premature stop codon is not spliced into the final transcript if L5 protein binds to the engineered RNA. On the other hand, if the premature stop codon is spliced into the final mRNA transcript then the mRNA is targeted as coding for an unnaturally short protein and is degraded by specialized cellular proteins **.
One can then put many protein-coding genes (~88% in Arabidopsis thaliana) under control of a specific protein using transgenic plants. The new project presented is to extend the ability of RNA to control gene regulation by binding small molecules instead of proteins. Some examples have already been shown to work in nature, but have not been applied to transgenic model plant organisms.
*Splice sites are spatially separated specific and non-specific sequences of nucleotide bases. The length of the splice sites separation varies greatly and allows for multiple proteins to be encoded from one gene depending on the combination of joined mRNA sequences. These splice sites are selectively joined together either with a splicozyme that is formed from the folding of that particular mRNA sequence or with help from proteins composed of multiple small nuclear RNA-protein complexes called ribonucleoproteins (snRNPs), pronounced 'snurps', collectively called splicesomes.
**Normally a mRNA transcipt that has a premature stop codon is recognized as junk by the cell and is degraded within the cell before translation in order to avoid expression of truncated proteins that could be toxic to the cell.
***What came first the chicken or the egg? Neither, they evolved together.
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