Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
24 Cards in this Set
- Front
- Back
What part of the snRNPS base pair to the splice sites? |
The RNA component of the snRNPs are what base pair to the splice sites because they have sequence complementary to these sites. The RNA component facilitates splicing. |
|
|
Do introns have a reading frame? |
No, only the exons have a reading frame because the introns are being spliced out and they don't have a reading frame. |
|
Alternative Splicing Review |
We don't need to use all exons, we can skip some and include different exons to make different versions of proteins that have different domains. With alternative splicing, we pick and choose which exons we remove. |
|
|
Alternative Exons |
We can have exons that are directly next to each other and we can either use both, or one of the other. |
|
|
We have a primary transcript that has exons 1,2,3,4. Exons 5 & 6 are alternative exons right next to each other. We can use both or one of the other. In the thyroid, we splice out exons 5 & 6. In the brain, we splice out exon 4. |
|
|
If you isolated mRNA from thyroid and brain and hybridized it to genomic DNA, how many loops would be found in each? |
They would both have 3 |
|
|
What is the advantage of having introns that need to be eliminated before the mRNA can be translated? |
- Alternative splicing allows a variety of related proteins to be synthesized from a single gene. - Main rationale used to explain the existence of introns is that it has allowed evolution to proceed at an increased pace. - Exons frequently encode different domains of a protein which can be combined via DNA rearrangements to generate new proteins relatively quickly (exon shuffling) - Because introns are so large, they are thought to be a “safe place” for DNA breaks, protecting the genome from mutations. -Prokaryotes do not have introns and don't undergo splicing! |
|
Example: Non-coding tRNAs are also spliced and undergo post-transcriptional modification |
tRNAs and rRNAs are the non-coding proteins. These RNAs aren't spliced, but they undergo a series of modifications. You can take the primary transcript of what is going to be a tRNA molecule, for example, and cleavage happens and some bases are added/removed. These modification occur before they become a mature tRNA. |
|
Example: Non-coding rRNAs are also spliced and undergo post-transcriptional modification |
With rRNA, they are synthesized as 1 RNA transcript. We have 3 different rRNA regions (16s, 23S & 5S) that are part of ribosome. It will be cut at specific sites to make mature rRNA. This shows how noncoding also undergo modifications other than the splicing out of introns. |
|
|
In eukaryotes, we have to export our mature mRNAs into the cytoplasm. Why? |
So they can be translated |
|
|
Ran |
Responsible for shuttling mRNAs in/out of the nucleus into the cytoplasm. |
|
|
GTPase |
An enzyme that hydrolyzes GTP (high energy molecule) to make GDP (lower energy molecule). It does this by hydrolyzing a phosphate group. When it is bound to GTP or GDP, it has a very different conformation and therefore has a different function. In most cases, Ran-GTP-bound form is going to be active form. The Ran-GDP-bound form is the inactive one. |
|
|
GTP & ATP difference |
Unlike ATP, which is normally associated with the transfer of energy, the GTPase is often associated with movement of a protein. The conformational change of Ran will change what it can/can't bind depending if it's GTP or GDP form. |
|
|
How do we import mRNAs from the cytoplasm into the nucleus? |
We do this with Ran-GTPase. Ran-GDP binds a separate protein called Importin. Importin helps carry the RNA into the nucleus. Once back inside the nucleus, Ran-GDP exchanges GDP for GTP, it no longer can bind that RNA or importin, but its free to bind exportin and another RNA. |
|
|
How do we export mRNAs from the nucleus to the cytoplasm? |
We do this with Ran-GTPase. When Ran is bound to its GTP form, it binds a protein called exportin. The exportin carries the RNA along with the Ran-GTPase, they enter a pore in the nucleus. Once outside into the cytoplasm, Ran-GTP hydrolyzes its GTP for GDP, it changes its conformation, releasing the RNA and the exportin carrier. |
|
RNA analysis is performed with which technique? |
QPCR based off RNA not DNA. PCR only works with DNA because it's double stranded, and doesn't work with RNA because it's single stranded. RNA is single stranded, but we do have a way of making DNA from RNA. |
|
|
Reverse Transcriptase (RT) |
Allows the conversion of RNA sequence into DNA. We utilize the fact that mRNAs all have a PolyA tail. We can isolate all the mRNAs from a cell based on the fact that they all have this tail. We can then make a probe or a primer to stick to the PolyA sequence, and pull out the mRNAs. Also, because of this PolyA, we can make a universal primer for synthesizing DNA from the RNA template for all mRNAs. |
|
RT Walkthrough |
We start out by annealing oligo dT primer (DNA primer made of Thymine), which will bind the PolyA tail. We can then use it to extend along the length of the RNA and make a DNA complement to the RNA molecule. To make a double stranded molecule, we can degrade the RNA component with RNAse (enzyme). Now we have a single stranded DNA copy and we could build up the other strand by using random hexamers. Then we need to ligate the random hexamers. Now we have a double stranded cDNA molecule to whatever our initial RNA sample. These cDNA molecules can be analyzed by QPCR. |
|
Quantitative Reverse Transcriptase-PCR (QPCR) |
You're quantifying the amount of cDNA from a sample. We use primers that are specific for the gene we are studying. We undergo PCR in a way that we can detect when we've made additional cDNA to have double strands. We have a fluorescent tag that will only light up when its bound to a double stranded molecule (cDNA). As we undergo more rounds of PCR, we generate more double stranded molecules. We will increase the level of fluorescence with each replication. Threshold in the pic is just something to compare to the fluorescent tag and we pay attention to something that passes the threshold sooner, because it will be more expressed. CT value is where gene passes the threshold. The more mRNA we have for a gene, the more cDNA we have for that same gene and the faster we will emit fluorescence. |
|
|
Patient A and D. The blue is our control (reference) gene. This is a gene that is very highly expressed. If someone has a high level of HepB, then it will barely surpass the reference gene just because the reference gene is so highly expressed. REMEMBER: The lower the CT number the faster it doubles and the more it is expressed, so that's why it's A and D. |
|
Microarray |
Like QPCR, this involves isolating mRNAs from a cell and reverse transcribing them into cDNA for analysis. When we do reverse transcription reaction, we can make it so the oligo-dT primer is labeled with either red or green fluorescence so that the entire cDNA population will be labeled red (cancer cell) and a non-cancer cell will be labeled green. |
|
|
When doing a microarray, what are we looking for? |
Looking for things that are different between samples, like something that is highly expressed vs something that is not expressed in another cell. Purpose is to find things that are interesting or things we could study. |
|
What do the yellow, green and red dots tell? |
They tell where the two cells we are comparing hybridize, meaning the expression of a gene is equal in the two. Where we see a green dot, that is exclusive to one cell. Where we see a red dot, that is something exclusive to another cell. |
|
|
Difference between Microarray and QPCR? |
The difference between Microarray and QPCR is you are not using specific primers for a gene, this is more "unbiased" and we detect any RNA. |
