Unit 3: Interphase Nucleus
NucleolusNucleoli are not bounded by a membrane. They are modified chromosomal loops that are transcriptionally active and involved in synthesis of rRNA.
Three types of components:1. DNA that is not actively synthesizing r-RNA. 2. RNA molecules in the process of rRNA synthesis 3. maturing ribosomal subunits.
The Nuclear PoreThere are lots of nuclear poresThey have an elaborate structure They are involved in the transport of RNAs and proteins.
What goes In/Out In: Histone moleculesPolymerases and other enzymes required for replication and regulation of replicationRibosomal proteins and other proteins (including snRNPs) that complex with newly formed transcripts.
Out: Ribosomal subunits m-RNA Protein complexes. Only fully processed and spliced transcripts are allowed out of the nucleus.
Transport into NucleusNucleoplasmin is a large protein with distinct head and tail regions. The tails can be separated from the heads by limited proteolysis. Colloidal gold is microscopic gold particles that can be made to a desired size and seen in the TEM.
Steps of Import:1. NLS protein combines with nuclear import receptor 2. The complex binds to a fibrils attached to the cytosol surface of the nucelar pore complex.3. The fibril then bends 4. The protein is transferred to the central component of the nuclear pore complex.5. The central component of the nuclear pore complex undergoes a conformational change that results in the protein being transferred to the nuclear side of the pore complex.
CHROMATIN and CHROMOSOMES Experimental studies
ChromatinThis schematic drawing shows the orders of chromatin packing that give rise to the highly condensed mitotic chromosome. Short region of DNA double helixBeads on a string form of chromatin30 nm chromatin fiber of packed nucleosomesSection of chromosome in an extended formCondensed section of chromosomeEntire mitotic chromosomeEach DNA molecule has been packaged into a mitotic chromosome that is 50,000 X shorter than its extended length
NucleosomesThe nucleosome consists of the core histone octamer surrounded by DNA wrapped around the outside of the proteins in approximately two turns per nucleosome. This interaction forms a 'beads on a string structure' Histone molecules: 2 - H2A2 - H2B2 - H32 - H4
What are Nucleases?Enzymes that cleave nucleic acids (RNA or DNA).Nucleases can be very specific,cutting the DNA or RNA between specific base pairs (targeting a specific nucleotide sequence).A mixture of different nucleases can be used to create random cuts.
Step 1 of DigestionChromatin is exposed to nuclease digestion.The timing of the digestion is such that not every stretch of linking DNA is digested.
Step 2The incomplete digestion of chromatin results in fragments of varying length, but in multiples of the length of one DNA/histone bead on a string.For example, if 100 bp was involved in one bead on a string, this digestion will produce fragments of roughly 100, 200, 300 bp etc.
Step 3Protein is removed to leave sections of naked DNA of varying length.
Step 4The DNA fragments are separated via electrophoresis on an agarose gel.The fragment lengths will be roughly multiples of the amount of DNA involved in one segment of the beads on a string model of histone-DNA packaging.
Characterization of NucleosomesA mixture of different nucleases will cleave any exposed section of DNA indiscriminately.Thus, a section of DNA that is protected by histones will not be digested. Nucleases can be used to determine the length of DNA involved in one nucleosome, because there is a repeating pattern of histone protection.Naked DNA, however, would produce a smear of many sized pieces of DNA until it is digested down to 1 base pair fragments.
Example:Lane 1: Nuclease digestion of naked DNA. Lane 2: No nuclease digestion.Lane 3: Nuclease digestion of DNA associated with histones.