Lec 9 Flashcards
Eukaryotic cells mostly have a
Circular nucleus
RBCs have no
Nucleus
Nuclear pores
Many pores exist and are very large
What enters the nucleus
Nucleotides
RNA Pols
Spliceosomes
Transcription factors
DNA Pols
Histones
Cdk/Cyclin proteins
What leaves the nucleus
mRNAs
tRNAs
Ribosomes
Nucleotides
Needed for RNA synthesis, DNA replication and DNA repair
Hypothesis for nucleotide movement
They are small enough to enter by diffusion
Experiment for nucleotide movement
Inject gold balls into cytosol of cell
Wait
See if the cells have entered using ultrathin sectioning + stain + TEM
Result of nucleotide movement
Diffusion is possible
Test of discovery
Observing a process
Test of necessity experiment
Removing something from a process and seeing if it still occurs
Test of sufficiency experiment
If you add something does a new process occur
NLS
nuclear localization signal
All proteins have an NLS
NLS discovery
Virus protein infects a cell and causes the host to burst with more virus cells
T-antigen amino acids 126-132 is
Necessary for nuclear localization
E coli experiment
Adding B-GAL to the T-antigen is sufficient for nuclear localization
Test of necessity is
Easier and should be done before test of sufficiency is possible
If you find something is not necessary there is no reason to do a sufficiency experiment
What is requires for nuclear import
An NLS (marks protein for being destined for the nucleus)
Importin (Import protein)
Importin
Binds to anything with an NLS and binds to nuclear pore to deliver cell. It lets go of cell once the import is done
Nuclear export needs
Needs NES (Nuclear Export Signal)
Exportin
Exportin
Associates with NES or RNA directly and nuclear pore
Same as importin mechanism but backwards
What drives transport in the right direction
Importins and exportins has a high affinity for the nuclear pore
Importins have a high affinity for the cargo in the cytosol but a low one in the nucleus, which is why they let go in the nucleus
The opposite is true for exportins
Ran
Ran-GTP is the active form
Ran-GDP is the inactive form
Ran-GTP has a high affinity for importins and exportins
Ran-GDP is in the cytosol and Ran-GTP is in the nucleus
When active Ran binds to the importin in the nucleus, importin lets go of its cargo and exits the nucleus along with Ram
When Ran binds to an exportin, it also binds to the cargo and leaves the cell, where it Ran becomes inactive and lets go of the exportin, exportin then lets go of its cargo and goes back to the nucleus alone
Affinity for the nucleus is due to
Ran
