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Lecture 14

Biology 2581B Lecture 14: Genes & Gene Expression 1

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Biology 2581B
L.Graham Smith

Lecture 14: Genes & Gene Expression 1 What is a gene? - The basic unit of biological information - Segment of DNA encoding a protein - Discrete region of a chromosome encoding a protein/RNA - These definitions are all WRONG! Example: look at the cox1 gene (in humans) - REMEMBER: the human nuclear genome is about 3 billion bp, human mitochondrial genome is about 16kb - Cox1 gene is about 1500bp long - RNA polymerase transcribes the double-stranded DNA into single-stranded mRNA - The mRNA gets translated into a protein that is 513 amino acids long - This gives us the protein called cytochrome C oxidase subunit 1 - If the mRNA = 900nt, we would get 299 amino acids o NOT 300 because the stop codon doesn’t code an amino acid!! (tricky) - This protein is involved in ETC, found in the Cu A centre (binuclear copper centre) - Cox1 is a part of a polycistronic RNA transcript - When you express Cox1, it shows up in a long RNA transcript that then gets processed o Essentially gets cleaved into individual mRNAs, some of which give you a protein (e.g. Cox1), others give you tRNAs or rRNAs - Not everything on the transcript encodes a protein!! - The cox1 gene itself is intact: the whole gene is there in one piece o Part of a polycistronic transcript o Encodes a protein o Continuous: the coding sequence begins and ends continuously - Let’s look at the same gene, but in a different species Cox1 in mushrooms - The Cox1 gene in mushrooms is about 20 times bigger than ours o Why is it so much bigger? It’s full of introns (19 introns, 20 exons!) - Those introns show up in the first mRNA that gets transcribed - Then the introns get spliced so you end up with a mature spliced mRNA that is more similar in length to the human Cox1 gene - In mitochondrial genomes, introns are slightly different than those in nuclear genomes o In mitochondrial introns, it’s much simpler; the intron will form an intricate secondary structure with loops and folds which allows it to easily be spliced out - The protein product is still the same - So the gene is 95% intronic! - The protein itself is only about 50 amino acids longer than ours Cox1 in Diplonema - This organism is a unicell - The Diplonema lineage represents the most abundant predator on our planet o Its mitochondrial genome is made of tons and tons of little circles - The Cox1 gene is not found on a single chromosome; the sequence for Cox1 is actually found on 9 different chromosomes! - When you want to express Cox1, you need to get 9 different transcripts, and then they all have to be stitched together at the mRNA level (need to make an intact transcript) in the right order to get the mature mRNA transcript o Then you can translate it into the functional protein - This is really weird, that 9 different chromosomes each have a small piece of the Cox1 gene - The way the genes get stitched together is called trans-splicing Trans-splicing - This is when exons located in distant regions/different strands/chromosomes are transcribed separately and then joined together - Shown in Diplonema - This process is pretty messy, not very well understood - We think that each of the exons has a little piece of an intron, then the two exons find each other while floating around o Introns will bind together by folding into their secondary structure, and that allows the two gene pieces to be spliced together Ribosomal slippage - Perkinsus is not an oyster, BUT it can parasitize oysters - The Cox1 gene in this organism is really weird - If you were to go along the DNA sequence in Perkinsus, you would find that it’s not in frame - The gene is found in frame, and mutations shift it to frame 2 and frame 3 o If there’s so many frameshift mutations, how can this be? - Sure enough, those frameshifts are conserved in the mature mRNA o We find 10 different frameshift mutations in the mRNA o This should make the gene non-functional - We then noticed that all frameshifts happened at certain motifs (e.g. AGGY or CCCCU) - People figured out that as the ribosome is reading the mRNA, it can actually skip/jump and correct the frame when it finds these motifs - So the frameshifts occur at a very conserved sequence motif, and the ribosome is evolved to recognize these motifs so it is able to jump - In the out-of-frame sequence, we would hit a stop codon - But in Perkinsus, the ribosome has evolved to be able to skip over the stop codon, and still transcribe the N amino acid Cox1 in Magnusiomyces capitatus - This is a parasitic fungus - In this case, the mRNA for the cox1 sequence shows the coding sequence in purple with some gaps in the middle that represent non-coding regions - We would think the black regions are introns - The only problem is that the black bits didn’t look like introns (no motif, which is classic signature of introns) - We also couldn’t figure out how the “introns” were be
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