C2006/F2402 '10 OUTLINE OF LECTURE #7
(c) 2010 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/09/2010 02:19 PM
Handouts (not on web):
7A & 7B -- nucleosome & chromatin
structure. Similar to Becker figs. 18-20 & 18-21.
For color versions of pictures on 7B
click here.
7C -- Sorting of Proteins (Overview).
NY Times Sunday Magazine of 2/22/09 has an article that you might find interesting about the case of a woman who may have a defect in her 'basement membranes.' Article is really about how doctors try to diagnose mystifying conditions.
I. Sorting of Proteins to their Proper Place: Overview See Handout 7C, or Becker fig. 22-14 (20-14) or Sadava 12.15 (12.14) -- terminology in Sadava is slightly different.
A. What determines the fate (final location) of each individual protein? The amino acid sequence of the protein itself. The ability of each protein to reach its proper destination is built into the protein. The presence (or absence) of localization signals in the amino acid sequence of the protein is the determining factor.
1. What's a localization signal: a group of amino acids acting as an "address" or "tag" directing the protein to a particular destination.
2. Terminology: The localization signal or "tag" is often called a localization sequence (LS) or patch.
a. LS -- if it consists of a continuous section in the peptide chain.
b. Patch -- if it consists of a contiguous section in the folded protein. (But AA are not next to each other in the unfolded chain.)
3. Use of tags: The localization sequence/patch directs the protein to the ER, nucleus, etc. Several different localization sequences, which are read sequentially, may be needed to direct a protein to its proper destination. If no "tag" or special sequence/patch is present at all, the protein remains in the (soluble) cytoplasm.
B. The Big Divide -- Attach to the ER or not?
1. Ribosomes start to make protein first. Translation starts first; then ribosome location (attachment to ER or not) is determined by the sequence of the protein being made.
2. Which ribosomes go to ER: If protein has the right "tag" (a localization signal called a signal peptide (SP) or signal sequence) -- the ribosomes attach to the ER, and protein enters ER as it is made.
3. Which ribosomes stay in cytoplasm: If there is no SP, ribosomes remain "free" in the cytoplasm -- they do not attach to ER or any other membrane.
C. Fate of proteins made on free ribosomes
1. Soluble Cytoplasm -- the default location for a soluble protein. If there are no "tags" at all, proteins stay in the cytoplasm.
2. Organelles. If proteins have the right "tags" they can be imported post-translationally (after synthesis) into organelles (nuclei, mito, chloro or peroxisomes) that are NOT part of the endomembrane system.
3. Terminology: "free" means not attached to a membrane. All ribosomes making protein, "free" or not, are attached to mRNA.
D. Fate of proteins made on attached ribosomes -- these become part of the endomembrane system and/or leave the cell. How they do this will be discussed in later lectures.
1. They enter the ER as they are made (co-translational import). Protein can
- Remain inserted in membrane -- become a transmembrane single or multipass protein (details later)
- Pass all the way across the membrane and end up in lumen of ER (as soluble protein or peripheral, membrane associated protein on lumen side)
2. Most proteins travel from ER to Golgi (a few may remain in ER).
3. Where do proteins go when they leave the Golgi?
b. To the plasma membrane -- Result is release of contents of vesicle outside cell and/or addition of material to cell membrane. Click here for animation #1 -- annotated & animation #2 -- larger but not annotated.
4. How do proteins travel from one part of EMS to another or to the plasma membrane? Vesicles carrying protein bud off ER, Golgi, etc., travel to another compartment (or plasma membrane) and fuse with target membrane.
a. Transmembrane proteins remain in the membrane. TM proteins move from membrane to membrane but always remain in the same orientation -- cytoplasmic domain remains in cytoplasm.
b. Soluble proteins stay soluble. Contents of lumen of compartment #1 → lumen of vesicle → lumen of next compartment or outside the cell.
Questions (to check your understanding of the
topology):
(1). Suppose domain X of a multipass integral membrane protein is on the
inside of the ER, sticking into the lumen of the ER. When a vesicle forms
off the ER, where will domain X be? Inside vesicle in lumen? On outside of
vesicle in cytoplasm?
(2). When vesicle fuses with Golgi, where will domain X be? In lumen of Golgi? Sticking out into cytoplasm?
II. How
Proteins Enter Nuclei -- Nuclear Pores and Traffic through Them
A. Pore structure -- Large complex structure that is hard to explain; will be demonstrated. See Becker fig. 18-27 & 28 and/or Sadava fig. 4.8 (4.9). Some links to pictures are below. Go to Google images for more pictures.
Two depictions of structure of a nuclear pore: Diagram #1; Diagram #2.
Diagram of how pores are located in the membrane.
Freeze fracture showing pores on surface of nucleus.
B. Pore Function
1. For relatively small molecules: Acts like (ungated, always open) channel for small molecules (may include small proteins). Transport is passive.
2. For big molecules: Acts as active transporter for big molecules. Features of active transport are below.
3. Transport is bidirectional -- mRNA, ribosomal subunits etc., must go out (see below) and proteins (as well as small molecules) must go in. (See Becker fig. 18-29; molecular details are in 18-30.) How things get out is not as well understood as how things get in.
4. Important Features of Protein Transport through Nuclear Pores
a. Transport requires a localization signal. Protein to be transported requires NLS (nuclear localization signal) to be transported into nucleus; NES (nuclear exit signal) on protein to be transported out.
b. Transport requires energy (GTP split).
c. 'Middle-man' protein required. Transport requires importin (to go in) or exportin (to go out).
d. Transport involves binding to pore protein.
Protein with NLS binds to "middle man" or "ferry proteins" (importins or exportins) which bind to pore protein.
Importin goes into the nucleus with the 'cargo' protein it is transporting in; exportin goes out with its cargo; both proteins then recycle.
Additional proteins (that we will ignore) are required as well. See Becker fig. 18-30 for a model.
Mechanism of transport through nuclear pores is similar, but not identical, in both directions.
e. Soluble and peripheral proteins are transported through pores. How do integral (transmembrane) proteins of the nuclear membrane reach their destination? Not through pores. TM proteins are probably made on the ER, and slide laterally into the outer & inner nuclear membranes (continuous with the ER). Once in place, TM proteins are anchored by binding to lamins or other internal nuclear proteins. Details of how TM proteins are inserted in membranes will be covered later.
To review nuclear transport, try problem 4-2.
III. Nuclear Structure & Role of nucleolus
A. Lamins A, B, C & Nuclear lamina
1. Lamins = IF's of nucleus (No MT or MF in nucleus)
2. Not to be confused with Laminin -- glycoprotein in the extracellular matrix (ECM) found primarily in the basal lamina
3. Form nuclear cortex or nuclear lamina = web supporting the nuclear envelope -- see Becker fig. 18-31(b). Similar function to ankryin/spectrin/actin web under plasma membrane -- provide support and points of attachment.
4. State of Phosphorylation and function change during cell cycle
a. Phosphorylated at G2 → M transition; Completely phosphorylated by prophase; due to rise in protein kinase activity; de-polymerize.
b. Dephosphorylated at anaphase, at start of M → G1 transition; due to rise in protein phosphatase activity; re-polymerize.
c. This is another example of using addition/removal of phosphate to control protein conformation & activity.
Kinases & phosphatases here are separate from their target proteins (as vs. case of Na+/K+ pump).
Same kinase or phosphatase can modify multiple target proteins at critical points in cell cycle. (Many proteins phosphorylated at entry to M and dephosphorylated at exit to G1.)
5. May help organize chromatin; may provide attachment sites for chromosomes and nuclear pore complexes.
Question: Should lamins have an NLS? NES? Should the LS be removed?
B. Nucleolus
1. Nucleolus has no membrane (see Becker fig. 18-33).
2. Nucleolus is site of rRNA synthesis and assembly of ribosomal subunits
a. Production of ribosomes requires extensive traffic in and out of nucleus
All transport in and out of nucleus is through nuclear pores.
Both RNA and protein must pass through pores.
Mechanism of transport through nuclear pores is similar, but not identical, in both directions.
Reminder: importin goes into the nucleus with the 'cargo' protein it is transporting in; exportin goes out with its cargo; both proteins then recycle. (See Becker 18-30.)
b. rRNA is made in the nucleolus (see Becker fig. 18-34) using RNA polymerase I (see below)
c. Ribosomal proteins are NOT made in the nucleolus.**
Ribo proteins are made in the cytoplasm, but imported into the nucleus for assembly into subunits.
Messenger RNA for ribosomal proteins is made in the nucleus using polymerase II (see below)and exported to the cytoplasm, as for any other mRNA.
After the mRNA is translated, the proteins are imported back into the nucleus for assembly with rRNA.
**Note: Becker says there are ribosomes translating mRNA in the nucleus. Maybe, maybe not. This finding is controversial -- the existence of nuclear ribosomes is not firmly established, so we are ignoring them. Ribo proteins are made in the cytoplasm in any case.
d. Ribosomal subunits are assembled in the nucleolus from ribosomal proteins (made in cytoplasm) and rRNA.
e. Completed ribosomal subunits are shipped out to the cytoplasm to be used in protein synthesis.
4. Nucleolus vs NOR (= nucleolar organizer region of DNA)
a. There are multiple copies of genes for rRNA; many copies are located in tandem. There may be one or multiple clusters of rRNA genes. Each cluster = one NOR.
b. Why many copies of rRNA genes? Multiple copies are needed because rRNA is made directly off the DNA -- no intermediate such as mRNA to allow for amplification. (Q to think about: Do you need multiple copies of the genes for tRNA?)
c. NOR = region of the DNA coding for rRNA = rDNA = genes for rRNA. Each NOR has multiple copies of the rRNA genes. Genome may have one or more NORs. NORs are permanent -- are part of DNA.
b. Nucleolus = structure seen in interphase; not in meiosis/mitosis.
Nucleolus forms during interphase at location of one or more NORs.
Several NORs can cluster together to form one nucleolus.
Nucleoli are only visible when NOR is being transcribed and ribosomes are being assembled.
c. Nucleolus (but not NOR) disappears during meiosis/mitosis since genes for rRNA inactive then (no rRNA made or ribosomal subunits assembled)
C. RNA polymerases & associated proteins
1. In Prokaryotes -- there is only one RNA polymerase.
2. In eukaryotes -- there are three RNA polymerases. Differ in sensitivity to drugs, location in nucleus, & what RNA's they make. (See Becker table. 21-1 for details; for reference only.)
a. RNA pol I → big ribosomal RNAs. (All ribo RNA but 5S.) This is the polymerase found in the nucleolus.
b. RNA pol II → mRNA (We'll focus primarily on this one when we discuss regulation of transcription.)
c. RNA pol III → small RNA's like tRNA, 5S RNA (small ribosomal RNA)
3. Eukaryotic Transcription factors (TFs)
a. Eukaryotic transcription requires additional proteins (TFs), not just RNA polymerase.
b. TFs are needed to allow polymerase to bind to DNA
c. There are two types of TFs (more details next time)
(1). basal (or general) -- required for transcription of all genes; are the same in all cells
(2). regulatory -- decrease or increase basal transcription (usually called repressors or activators respectively). Are different in different cell types.
To review nuclear structure & transport into nuclei, try problems 4-2, 4-6 parts A & B, and 4-7, parts A & C.
D. Chromatin. DNA in eukaryotes is not "naked" as it is in bacteria. It is in the form of
chromatin = DNA complexed with proteins. So how do polymerases and TFs get to the DNA and how do they manage to carry
out transcription with all those proteins stuck to the DNA? What
actually turns transcription on and off? To answer
these questions, we need to look first at chromatin structure.
IV. General Chromatin Structure -- Euchromatin & Heterochromatin
A. Composition of chromatin = DNA + associated proteins
1. Associated proteins are mostly histones (small, basic; more details to follow)
2. Associated proteins includes some (nonhistone) regulatory proteins
B. States of chromatin (See handout 7A)
1. In all states, DNA has proteins attached
2. Usually differences are due to different states of folding after histones added, not removal of histones (there are some exceptions which will be discussed later).
3. Two Types of Chromatin -- Two basic states of chromatin are visible in the light microscope
a. Heterochromatin or heterochromatic chromatin/DNA
(1). What is it? Chromatin that is darkly stained, & relatively condensed (tighter),
(2). Heterochromatin is genetically inactive (not transcribed)
(3). Two kinds of (interphase) heterochromatin
(a). Constitutive heterochromatin -- always heterochromatic in interphase (ex: chromatin at centromeres, telomeres). Usually repeating in sequence, non coding.
(b). Facultative heterochromatin-- sometimes heterochromatic in interphase, sometimes not (depends on tissue, time etc.). Example: inactive X. Same X is not inactive in all cells. Whichever X is inactive (forms a Barr body) is heterochromatic.
(4). Most of the DNA that is inactive during interphase (in a particular cell type) is NOT heterochromatic. See (3) below.
(5). All chromatin is heterochromatic (tightly condensed) during mitosis & meiosis.
(6). All chromatin, both heterochromatin & euchromatin (see below) is replicated in S. Heterochromatic DNA is not genetically active (not transcribed), but it is replicated.
b. Euchromatin or euchromatic chromatin/DNA
(1). What is it? Chromatin that stains more lightly, is less condensed (looser).
(2). Euchromatin is capable of genetic activity (transcription) as vs. heterochromatin. Normal state of most DNA during interphase. Transcribable, but not necessarily being transcribed now.
(3). Most interphase DNA is euchromatic, whether it is transcribed or not. DNA must be euchromatic to be active, but not all euchromatin is active.
(4). Not all euchromatin is folded equally tightly
(a). Tighter vs looser. Euchromatin is often divided into several distinct states of folding, although tightness of folding is probably really continuous from relatively loose ↔ relatively tight.
(b). Different states of (eu)chromatin look about the same in the light microscope. Therefore indirect methods (such as DNase treatment) are necessary to test state of folding. Details are below.
(c). Correlation between folding and function. In general, more active (transcribed) chromatin is looser, but the situation is complex -- details to follow.
V.
Histones & Details of Chromatin Structure
A. Structure of Chromatin -- how are proteins and DNA arranged? How does all that DNA fit into a tiny cell? The evidence.
1. Appearance in EM -- low salt ("beads on a string" appearance) vs physiological salt (30 nm fiber). See Becker fig. 18-17 or Alberts (Or go to PubMed at http://www.ncbi.nlm.nih.gov/sites/entrez , click on books on upper right, and use the search term box. )
2. Results of treatment with DNase
a. Specificity of this DNase. Enzyme used here is microccocal nuclease; cuts DNA in areas that are most exposed or least protected. Where it cuts depends on local chromatin structure, not on the base sequence of the DNA. (Restriction enzymes are specific for certain sequences; this enzyme is not.)
b. Procedure: Treat chromatin (not DNA, but DNA with associated proteins!) with a microccocal nuclease; then remove protein and isolate the DNA, then run DNA on a gel.
c. Results & Conclusions:
(1). If you use a little nuclease: A little digestion with nuclease → 200 Base Pair (BP) "ladder" = sequences of multiples of 200 BP on gel. Implies repeating structure ("bead") with about 200 BP per repeat; DNA exposed (so easily cut) about once per 200 BP. (See Becker fig. 18-18 & 18-19.)
(2). If you use a lot of nuclease: More treatment with nuclease → resistant core of around 145 BP. Implies part of 200 BP repeat is relatively protected in/on core of "bead"; rest goes between beads and is more exposed.
3. Histones
a. 5 types: H2A, H2B (slightly lys rich), H3, H4 (arg rich) & H1 (lys rich). All relatively small, basic proteins.
b. All histones are highly conserved evolutionarily (this implies histones carry out a critical function that depends on a particular structure -- can't change structure much without ruining function).
b. How much per 200 BP of DNA? 2 molecules each of H2A, H2B, H3, H4 plus one molecule of H1.
c. Low salt removes H1. (It's on outside of bead; more easily removed.)
B. Model for Chromatin (Beads on string level) -- nucleosomes (See handout 7A and Becker fig. 18-20 or Sadava fig. 9.8 (9.6)). Where is the protein relative to the DNA?
1. Octamer of 2 each of H2A, H2B, H3, H4 (+ some DNA) = one bead.
2. DNA wound 2X around (on outside of) each octamer -- protects core. For more pictures and info on nucleosomes, click here.
3. Linker DNA between cores = 50-60 BP; most exposed & most sensitive to nuclease used above.
4. H1 is on outside of DNA/octamer
5. Nucleosome = repeating unit = 200 BP DNA + octamer (H1 optional).
C. Nucleosomes & Higher Levels of Structure (requires H1) -- how does chromatin fold up? See 7B or books for detailed pictures. (Becker fig. 18-21 or Sadava fig. 9.8 (9.6) or click here.
) Stages of folding are as follows (details in table below):1. Nucleosomes. DNA + histones form a chain of nucleosomes; about 1/7 original length of DNA (see table below). Also called a "10 nm fiber."
2. 30 nm fiber. Chain of nucleosomes folds back on itself (supercoils) forming 30 nm fiber (sometimes called solenoid). Exact structure unclear. Fiber is about 3 beads across; 6 beads/turn = 1/42 length of DNA. Need tails (of histones) and H1 to form 30nm fiber.
3. Loops. 30 nm fiber forms loops about 300nm in diameter (1/750 orig. length). Different sections may be tighter or looser. Individual loops are stretched out (probably to beads-on-a-string stage) when actually transcribed.
4. Higher Orders of Folding. Looped structure folds further → → heterochromatin (not transcribable)
a. Chromatids: Folds back on self to form structures/fibers about 700 nm across (per chromatid)
b. At metaphase = tightest = 1/15,000-1/20,000 orig. length (Chromosome is 4-5 microns long but contains 75 mm of double helical DNA)
D. Summary of States of Folding -- compare to handout 7B.
|
Structure |
Compaction relative to previous |
Packing Ratio* |
Diameter |
H1 Needed? |
|
DNA |
none |
1 |
2 nm |
-- |
|
Nucleosomes -- beads on a string |
7X |
7 (1/7th length of DNA) |
10-11 nm |
no |
|
30 nm fiber |
6X |
42 |
30 nm |
yes |
|
loops |
15-20X |
750 |
300 nm |
yes |
|
heterochromatin (metaphase) |
20X |
15-20,000 |
700 nm (per chromatid) |
yes |
* Packing ratio = length of DNA or DNA/protein complex relative to original length of DNA. As packing ratio increases, chromatin fibers get shorter and wider.
E. Modifications of histones -- helps tighten up or loosen chromatin
