Lecture 22

Front 1

How would you describe the major difference between basal and activated transcription?

Back 1

Basal uses only the general transcription factors to initiate transcription, but is not acting at its best possible performance/ rate.

Activated transcription accelerates the binding of the GTFs to the DNA using modular transcription activators which are a domain associated with the function of binding the GTFs. This is much more efficient.

Front 2

How do cells know where to structurally assemble different body parts?

Back 2

Through homeodomain proteins. Recognition helices interact with the major groove and bind to specific DNA sequences. These specific sequences is a certain binding domain which is characteristic of an entire class. Cells know which type of structure they should code for based on information that is proximal, distal, ventral etc. An example of things going wrong is antennapedia mutations which is homeotic.

Front 3

What are zinc finger binding domains and what are the 3 types?

Back 3

Zinc finger domains are structures which interact with nucleotides to form zinc ions with cystines and histidines to form finger like projections by folding that interact with DNA.

C2H2 - 2 cys, 2 his, binds to DNA as monomers

C4- 4 cys, nucleohormone receptors, bind to DNA as dimers

C6 - 6 cys, corresponds to Gal4, the cystine metal ligands coordinately bind 2 Zn ions.

Front 4

What are similarities and differences between Leucine Zipper proteins and Helix Loop Helix proteins?

Back 4

Both are 2 proteins that interact through a hydrophobic interface to form homo and hetero dimers when binding to DNA through hydrophobic amino acids in the C-terminal region of the DNA binding domain. Homo and Hetero dimers decide how the proteins function upstream to change transcription activation through changes in conformation.

LZ has an extended alpha helix that binds the DNA's major groove, where HLH is coupled by a loop which allows for other helices to interact with the DNA.

Front 5

What is one way to tell the DNA sequence that proteins are binding to?

Back 5

DNAse footprinting. Uses idea that proteins will protect sequence from DNAse degradation.

1. Figure out how to get one cut per DNA molecule.

2. Radio-label probe at 5 prime end

3. Subject the single stranded oligonucleotide to phosphorylation then form duplex using complementary nucleotides.

4. Combine the radiolabelled DNA with protein mixture, then bring in DNAse to make cuts.

5. Denature molecule through polyacrylimide gel to see different molecule sizes then form ladder.

6. By adding the protein binding sequence, DNAse can't cut in certain places, leaving windows in the ladder.

7. We can see the importance of certain nucleotides and which ones are being protected.

Front 6

Why is ChIP-seq a better alternative to DNAse footprinting?

Back 6

It allows us to understand all genes in the genome at once using antibodies to see which sites are being bound, where DNAse footprinting requires finding a specific location which can be a long and expensive process.

First, we take the interested protein, shear it to get chromatin, then interact it with antibody to the interested protein. The antibody recognizes the protein then we can pull down the entire mixture using beads with Protein A to interact with antibody. The precipitate, wash and sequence DNA using Next gen. The DNA binding elements can then be determined using statistical analysis. There will be a common genome binding site.

Front 7

How do combinatorial possibilities extend the potential for diversified gene regulation?

Back 7

Heterodimerization gives combinatory power and controls the diversity of transcriptional responses.

Also, transcription factors of unrelated classes can bind cooperatively to be much more efficient, ie NFAT and AP1.

Flower organs are an example of this. Each specified organ is a product of combined effects of transcriptional factors through the MADS box class transcriptional binding factors.

Front 8

Why is the carboxy terminal domain of Pol II so important?

Back 8

Differentiates the large subunit from others and gives rise to RNA processing by removing introns. When it is phosphorylates at Serine 5 of YSPTSTPS by TFIIH, the promoter is cleared and elongation happens. The CTD is also critical for capping, which is enhanced by serine 5 phosphorylation, splicing, polyadenylation and export.

Front 9

Why is the phosphorylation at serine 5 such an important step?

Back 9

It enhances the interaction of the capping enzyme with emerging Pre-mRNA by adding 7 methylgluamate CAP to make 5 prime to 5 prime linkage. The 2 prime hydroxyl is also methylated on its first base and second base in vertebrates. The cap protects, is important for nuclear export and recognition by transcription factors.

Front 10

How was intron splicing discovered?

Back 10

By visualisation of the hybridization experiments of the adenovirus hexon gene. There was the formation of a DNA-RNA hybrid with loops of the intron sequences. This was also compared to the discrepancy between genome and mRNA length.

Front 11

What are the conserved regions of the introns and exons?

Back 11

The intron exon borders are highly conserved. They are almost always 5' GU and 3' AG. The adenosine branch point which is found just upstream of the pyrimidine rich region is also necessary for splicing.

Front 12

What is the purpose of small nuclear RNAs?

Back 12

They give rise to snRNPs through the U1, U2, U4, U5, U6 subunits. U1 is for the 5' border and U2 interactions causes branch point to bulge out and interact by being more accessible. There is Watson Crick base pairing at the U1 5' splice site, demonstrated by mutation analysis.