Exam 2
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DNA to RNA, uses RNA polymerase to make RNA polymers. Initiation, elongation, termination.
Why do prokaryotes need txn
Prokaryotes change gene expression often to break down and process their surroundings. Different enzymes need activated based on substance being consumed.
Prokaryotic promoters and regions
TT-35GACAT, TAT-10AAT
RNA polymerase I roles in txn
Core enzyme plus sigma factor is holoenzyme. Alpha, alpha(2 total), beta, B’, omega. Sigma factor binds to the promoter and the core enzyme to initiate txn. Core enzyme is used in elongation phase. Breaks Hydrogen bonds of DNA, not sugar phosphate backbone, and creates new SP backbone with RNA.
During elongation, RNA poly.
Releases sigma factor, core continues to transcribe. Only ~15 base pairs unwound at a time. Transcribed DNA is rewound and reforms bonds.
Termination in prok.
Uses either a Ro protein to stop, or more commonly Ro-independent termination where repeating sequences of AGCCC and UGCCC bond to make a hairpin loop secondary structure. The hairpin forces the release of core RNA poly. from DNA to end the process.
Eukaryotic Nucleus contains
Nuclear membrane (2 phospholipid bilayers), Nuclear Pore Complex, Nuclear Lamina
Outer nuclear membrane is continuous with
ER membrane, interacts with cytoskeleton and ribosomes.
Inner membrane
Interacts with the nuclear lamina and chromatin DNA in the nucleus. Contains lamin B receptors, and emerin. LBRs interact with the lamina, emerin interacts with chromatin.
Nuclear pore structure
Cytoplasmic filaments on outer end, channel in the middle, nuclear ring towards the nucleus on the inside, and nuclear basket on the inside of the nucleus. Made of 150 nucleoporin proteins. Structural polarity present, with differential head and tail.
Nuclear Lamina
Network of proteins on the inside of the nucleus along the inner membrane. Made of inner filaments lamin proteins. Disruption in lamins can disrupt nuclear shape overall.
Nuclear Transport
Histone proteins need to enter nucleus to make chromatin. All proteins for DNA replication and Txn must enter the NPC. Essential for any substance moving in or out of the nucleus.
Steps of nuclear transport
Example Erk trying to enter nucleus. Protein bind to Erk that contains a nuclear localization signal in its DNA sequence. NLS binds to importin and whole structure moves through NPC. Simultaneously, monomeric G protein Ran-GDP enters nucleus through passive diffusion. Ran-GDP uses its GEF inside the nucleus to become Ran-GTP. Ran-GTP then binds to the importin attached to the NLS, forcing it to change conformation and split off of Erk. Erk released in nucleus with NLS.
Export cycle of nuclear transport (Ran-GTP and importin portion)
Ran-GTP diffuses out of nucleus passively while still attached to importin. In cytoplasm, a GAP removes its phosphate group to make Ran-GDP, splitting off the importin. Both substances recycle into the import process.
mRNA leaves the nucleus by
binding to exporter protein exportin, which carries it out of the nucleus
Polymerases used in Eukaryotic txn
RNA polymerases I, II, III.
RNA polymerase I
makes most ribosomal RNA, 45s
RNA polymerase II
makes mRNA
RNA polymerase III
makes tRNA and one rRNA, 5s
Eukaryotic Promoters
Most prominent TAT-25AAA, also BRE sequence, CAT box, GC box.
Eukaryotes contain ___ for txn
Assisting proteins known as txn factor proteins. Examples are the TFII(letter) proteins.
TFIID in txn
uses TATA-binding proteins to bind to TATAAA box at -25 site.
After TFIID bound to promoter
TFIIA, then TFIIB bind to TFIID to create a txn complex. TFIIF and RNA poly II then bind to D,A,B, and DNA
After Txn complex receives TFIIF and RNA poly II
TFIIE and TFIIH bind to DABF, RNA polymerase II, all bound to DNA. TFIIH is a helicase AND a kinase, so it unwinds DNA near the +1 site, and phosphorylates RNA polymerase II.