Ls3 - Lecture 12

1. Eukaryotic transcription happens in the context of nucleosomes/chromatin. Prokaryotes don't have a nucleus and their DNA is part of the cytoplasm. They don't have chromatin, heterochromatin or other remodeling factors.

2. The level of regulation for eukaryotes is much higher.

1. Both promoters have AT rich regions and a +1 site. They also both have specific factors that bind the promoter region.

- Prokaryotes: -10,-35 box bound by sigma factor

- Eukaryotes: TATA box bound by TATA binding protein

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The RNA that is found in the nucleus. Since RNA is spliced, it can be found within many different lengths within the nucleus (this is different than what is found in the cytoplasm). RNA are not exported from the nucleus to the cytoplasm until they are fully spliced & have their 5' cap & 3' PolyA tail. Therefore, RNA located in cytoplasm are different from RNA in the nucleus.

It becomes shorter. So hnRNA is longer than mRNA on average. RNA starts out longer but by the time it gets to the cytoplasm, that same RNA is a lot shorter.

In addition, distinct intervening sequences are removed in the maturation process, and is shown by the discrete bands on the Northern Blot instead of a smear.

D. Intervening sequences are removed in big chunks, not continuously or would have a long smear. The probe will still anneal because it binds to the exon region & that's where info is retained. Introns are spliced, not exons.

mRNA is shorter than the original RNA transcript or the hnRNA. DNA is the original and the RNA transcript is what will be translated into protein, it's much smaller. You can hybridize mature RNA to the DNA sequence from which it came from because it's complementary from which it's transcribed from. Hybridizing them will yield bubbles or loops, which represent intronic sequences. There is no sequence complementary on the introns.

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Exons are often very small in comparison to intronic sequences. When you take exons all together, you make a very short mRNA. Some can also be very large. They very in size, as well as genes, ranging from as little as 2000bp to 200,000bp.

Within exons their are different variations of exons we can keep. Occasionally we will have intronic sequences that are supposed to be retained, and we'd splice an exon instead. The point: splicing can make a variety of stuff from a single genomic sequence.

Here we are showing that we have our DNA (with promoter, USE, exons, introns etc). The primary RNA transcript (hnRNA) is shown, the introns are removed and we are left with the coding region of that mRNA.

The mRNA will have regions upstream/downstream from the coding region (called 5' UTR & 3' UTR). Exon 1 is not necessarily the start of the coding region (it rarely is). The coding region is the specific part that will become a protein, however there are regions on either side of it called UTRs. These regions aren't actually translated. They serve as regulatory functions for regulating translation. 5' & 3' can serve as the binding sites for other factors that control mRNA stability or the rate of translation of that mRNA.

So we go from DNA -> hnRNA (remove introns to get final) -> mRNA.

For each of those regions where we have an intron in between two exons, we have specific sequences that are highly conserved (if these sequences mutated, you could affect splicing of introns/exons).

For each intron, we have something called a branch point A. It's always an A and somewhere near the middle of the intron.

We have a 5' Splice site which is usually a GU or AU. We also have a 3' Splice site which is usually a AG or AC.

They facilitate splicing reaction and occur through transesterification reactions.

Splicing requires two transesterification reactions. In the very beginning of splicing, we have one factor that will bind within the branch point & make the A bulge in a way that it's 2' OH can act as a nucleophile. It will attack the phosphate group that is attached to the 5' Splice site within the intron. By doing this, we've joined the intronic sequence together and left the exon hanging with a 3' OH. This 3' OH was connected to the phosphate group of the 1st nucleotide in the intron. This 3' OH can now act as a nucleophile for attacking the phosphate group on the 3' end of the intron. This joins the two exon sequences leaving every last base pair that was part of the intron in what we call an intron lariat (loop). The lariat can just be degraded by the cell but the exons are retained, because the whole point was to rid the intron. This process happens to all introns

snRNPS are enzymes composed of a complex of RNA and Protein. They are located in the nucleus

Their RNA sequence.

Example: U1 snRNP binds region at 5' splice site because it has a complementary sequence. U2 snRNP binds branch point A's complentary sequence. Other steps occur, but together, these proteins interact with each other while bound to the RNA in a way to help facilitate those transesterification reactions (because transesterification doesn't happen alone, these enzymes make it possible).

That would block splicing of the intron at that point.

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B. (The lariat isn't formed last. It's formed, then removed!).

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Here we have a primary RNA transcript (light green = introns, multicolored = exons). In the top, we have an mRNA that can be spliced into different sequences that can be made into different proteins. We can see that for the top one, we've included exons 1, 2, 3 and 5. On the bottom, we've included exons 1, 2, 4 and 5. When we do a splicing reaction, we can splice out an exon with two intronic sequence as 1 giant lariat. That will allow us to have more diversity in RNAs and proteins that we make. OFten, the exons that are alternatively spliced encode for a particular domain so we have have 2 proteins that have a similar function. One has a region that targets it to the plasma membrane, another doesn't have that. (So plasma membrane and cytoplasm version of protein).

Exons associate with 1 or more domains. We can have exon 1 make domain 1, and exon 2 make domain 2. Both of these synthesize as 1 transcript. Basically we get more diversity in proteins.