Indirect Protein Sequencing Via Geneomic Analyses - Video Tutorials & Practice Problems
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Indirect Protein Sequencing Via Genomic Analyses
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Video transcript
Hey, guys. In this video, we're gonna talk about indirect protein sequencing via genomic analyses. So up until this point in our course, we've only focused on direct protein sequencing methods such as tandem mass spectrometry or Edmond degradation. Now direct protein sequencing is used on already extracted or isolated proteins. And direct protein sequencing is able to directly identify the sequence of unknown proteins in a sample. However, direct protein sequencing does not account for how biochemists obtain most of their protein sequencing data. And so most of the protein sequencing data is actually derived indirectly from genomic analyses or translating the nucleotide sequences of genes into amino acid sequences. And so this brings up the question, why is most of the protein sequencing data obtained via genomic analyses? Why would we obtain most of our protein sequencing data this way? Well, it turns out that it actually saves a boatload of time. It saves a lot of time. Working with DNA is actually easier than working with proteins in a lab and that's because we know that proteins are really sensitive to lots of conditions, and they can be pretty easily denatured if the temperature is off or if the pH is different. And DNA is more resistant to, essentially decomposing and, breaking apart. And so because DNA is more stable, it's easier to work with, and so that allows us to essentially work with DNA faster. And it turns out that DNA sequencing is actually significantly faster, cheaper, and more efficient and informative than direct protein sequencing since direct protein sequencing only allows us to obtain the amino acid sequence, but DNA sequencing allows us to obtain the nucleotide sequence. And then from that nucleotide sequence, we can derive derive the amino acid sequence using the genetic code. And so essentially, overall, genomic analyses allows us to collect more data and more protein sequencing data faster. And so that break that begs the question, why do we even need direct protein sequencing if genomic analysis is the best way that allows us to obtain more protein sequencing data faster? Why do we even need direct protein sequencing if indirect genomic analysis is the best at that? Well, it turns out that we can't just scrap direct protein sequencing because direct protein sequencing has its own sets of advantages. And some of those advantages include the fact that genomic analyses are not able to identify an unknown protein sample on its own. And so, because it cannot do this, that's something that direct protein sequencing is easily able to do. And that's because when we're working with genomic analyses, we're going to need a DNA sample. And so, if we only have an unknown protein or just protein, then we're not able to perform genomic analyses on these proteins. So, it's not, that's not a good thing about genomic analyses. Now, in addition to that, unlike genomic analyses, direct protein sequencing via tandem mass spectrometry can actually reveal chemically modified amino acid residues. And that allows us to identify, essentially, proteins that are genes And so genomic analyses does not reveal chemically modified amino acid residues, but direct protein sequencing can. So that's another advantage of direct protein sequencing and another reason for why we can't just scrap all of the direct protein sequencing techniques. So the rest of this video here is going to refresh our memories on how the genetic code works, which allows us to perform genomic analyses. So recall from our previous videos that the genetic code actually reveals the connection between the codons of nucleic acids and the amino acids of proteins. And so in our example below, we're going to use the genetic code to reveal the peptide sequence in the example shown over here on the right. And so, what you'll see is on the left here we have the genetic code. And recall that the genetic code is essentially reading the codon of the mRNA, and the codons have 3 nucleotides. So with this genetic code, we have the first base of the codon on the left, we have the second base of the codon, so the second base of the codon, on the top here, and then we have the third base of the codon over here on the right. And so recall that the first base of the codon limits us to one particular row here. The second base of the codon limits us to one particular column. And then the 3rd particular codon limits us to a specific position in a box. And so, what you'll see here is that we have a DNA coding sequence that's provided, and you can see that it has a 5 prime end and a 3 prime end. And so we know that this DNA coding sequence can be converted into an mRNA sequence through the process of transcription that's shown here, represented by this arrow. And mRNA sequence is going to be exactly the same as the DNA coding sequence up above, except the fact that all of the threonines, are going to be converted into U's, or uracils, because mRNA only has uracils. And so these two threonines here are going to be converted into uracils in our RNA sequence. And so, now that we have our mRNA sequence, we know that the genetic code breaks down and reads the mRNA sequence in codons, which are sets of 3 nucleotides. So our first codon are these first three nucleotides, AUG. And so again, the first base of our codon is A. And so because it's A, it limits us to this column. I'm sorry, this row. The second base of our codon is u, so we can see that here, u. And so in the second base of our codon, it limits us to one particular column. So the overlap between these two is this box right here. And then the 3rd codon is, I'm sorry, the 3rd base of our codon is g, and so that limits us to this particular position within the box, which is a u g. An a u g codon corresponds with a methionine amino acid residue, which is why we have methionine as our first residue on the n terminal end of our peptide. So moving on to our next codon, we have g c u. And so g c u corresponds with this, first residue here in this column, I'm sorry, this row. Then we have c, which limits us to this column. So now we're in this box. And then u limits us to this one particular position, g c u, which is an alanine amino acid residue. So over here, we can put an a for alanine in that position. And so essentially what we can do is continue through this process here and move on to our next codon. So the next codon is g g c, and g g c, g is here in this row. G, the second one is G, so that limits us to this column, so now we're in this box. And then C here limits us to a GGC, which is glycine, so glycine is our next residue. And now you guys are probably remembering how this works here, and so what we can do is fill out the rest of these codons here. So we have, after g g c, we have cgg, then we have a g c, and then last but not least, we have a, a, a. And so CGG corresponds with an arginine, so this is an arginine, CGG. And then, AGC corresponds with a serine, and then of course AAA corresponds with a lysine. And so, what we can see is that the amino acid sequence of our peptide is actually revealed through genomic analysis. We obtain the DNA sequence and we sequence that DNA, And then, through the process of transcription and translation, the genetic code, we are able to obtain the sequence of our peptide. And so this is an indirect method to be able to sequence our peptides. And that's exactly how, indirect sequencing via genomic analyses works. And so in our next couple of videos, we'll be able to get some practice utilizing the genetic code and indirect protein sequencing. So I'll see you guys in those practice videos.
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Problem
Problem
Use the genetic code above & the coding DNA sequence below to determine the protein sequence.
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Problem
Problem
Suppose the sequence below is a template DNA sequence. What is the corresponding protein sequence?
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5m
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Problem
Problem
Even when the sequence of nucleotides for a gene is available and genomic analyses can be performed, direct chemical techniques on the physical protein are still required to determine:
A
The molecular weight of a simple protein.
B
The N-terminal amino acid residue.
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The total number of amino acid residues in the protein.