C2006/F2402 '04 OUTLINE OF LECTURE #8

I. Peroxisomes -- summary of structure, function and synthesis.  For more details see Becker  pp. 358-362.

    A. Structure -- comparison to mitochondria and lysosomes (See table in previous lecture.)

    B. Major Function = detoxification (in animal cells). See Becker for other roles, esp. in other organisms.

    C. How do Proteins (& phosopholipids) get into Peroxisomes?

• Peroxisomes have no DNA, but multiply by growth and division.

• Peroxisomes are not part of endomembrane system --  all the proteins of the matrix and probably all the proteins of the membrane (see note) are made on cytoplasmic free ribosomes and imported post-translationally.

•  Localization signals for best known peroxisomal enzymes are on COOH end of protein. (Some perox. proteins have the signal elsewhere.)

• Mechanism of import is not well understood -- matrix proteins may enter without unfolding. Entry requires ATP.

• Some phospholipids of peroxisomal membrane are made on the ER. Carried to peroxisome by transport/exchange proteins.
   

• Note: Some recent experiments raise the possibility that a few proteins of the peroxisomal membrane may originate in the ER, and that peroxisomes may sometimes arise de novo (as vs. by splitting of pre-existing peroxisomes). However, most of the experiments to date support the generally accepted view that peroxisomes do not arise de novo and that all protein components are made on free ribosomes.

To review how proteins enter mitochondria and peroxisomes, try problems 3-6 & 3-7.

II. How Proteins Enter Nuclei -- Nuclear Pores and Traffic through Them

    A. Pore structure -- hard to explain; will be demonstrated. See Becker fig. 16-27 &16-28 and/or Purves fig. 4.8 (4.10)

    B. Pore Function

1. Acts like (ungated, always open) channel for small molecules (may include small proteins).

2. Acts as active transporter for big molecules. Features of active transport:

a. Transport requires energy (GTP split)

b. Requires NLS (nuclear localization signal) on protein transported into nucleus; signal not removed (may be needed again after mitosis to re-localize protein)

c. Involves binding to pore protein

d. Probably works like SRP/SP/Docking protein system with three components -- Protein with NLS binds to "middle man"  or "ferry proteins" (importins) which bind to pore. See Becker fig. 16-30 for a model.

3. Transport is bidirectional -- mRNA, ribosomal subunits etc., must go out (see below) and proteins must go in. (See Becker fig. 16-29) How things get out not as well understood as how things get in.

To review nuclear transport, try problem 4-2.

III. Summary of Transport, Localization signals, etc.

Issues to keep in mind --  (See bottom of handout 6A or table below)

1. What type of localization signal (or localization sequence = LS) required? Where is it?

2. Is signal removed after membrane is crossed?

3. How many membranes must be crossed?

4. What is signal or sequence called?

^#If protein is going to pass entirely into lumen of ER, SP is always on amino end. If protein is going to insert into the ER membrane,  SP can be in middle and lock the protein into the membrane. 

 * This is for transport from cytoplasm into the matrix. Additional signals are needed to be retained in (or re-enter) the inner membrane, outer membrane, and/or intra-membrane space. Transport into chloroplasts is assumed to be similar.

** Depends how you count. Have to get across nuclear envelope consisting of two membranes, but passage is through pores and both membranes are crossed together. Answer could be 0, 1, or 2 depending on how you look at it.

To review localization and transport, try problems 3-14 & 3-15. (Skip protein (5) for now.)   By this point you should be able to do all the problems in Set 3 (except for protein (5) in 14 & 15) .

IV. Nuclear Structure

    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 -- a glycoprotein in the extracellular matrix (ECM) found primarily in the basal lamina

3. Form nuclear cortex or lamina = web supporting the nuclear envelope -- see Becker fig. 16-31 (b). Similar function to ankryin/spectrin/actin web under plasma membrane.

4. State of Phosphorylation and function change during cell cycle

a. Phosphorylated at G2 --> M (prophase); de-polymerize; due to rise in protein kinase activity

b. Dephosphorylated at M --> G1 (anaphase); re-polymerize; due to rise in protein phosphatase activity

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.

5. May help organize chromatin; keep it proper distance from pores

    B. Nucleolus

1. Nucleolus has no membrane (see Becker fig. 16-32). 

2. Nucleolus is site of rRNA synthesis and assembly of ribosomal subunits -- production of ribosomes requires extensive traffic in and out of nucleus

a. rRNA is made in the nucleolus (see Becker fig. 16-33) using polymerase I (see below)

b. 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 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.

c. Ribosomal subunits are assembled in the nucleolus from ribosomal proteins (made in cytoplasm) and rRNA.. 

d. Completed ribosomal subunits are shipped out to the cytoplasm to be used in protein synthesis. Export out of the nucleus through the pores requires an NES (nuclear export signal). NES allows transport out of nucleus; NLS allows transport in to nucleus. Mechanism of transport through nuclear pores is thought to be similar, but not identical, in both directions.

3. Nucleolus (but not NOR, see below) disappears during meiosis/mitosis since genes for rRNA inactive then (no rRNA made or ribosomal subunits assembled)

4. Relationship of nucleolus to NOR (= nucleolar organizer region of DNA)

a. NOR =  DNA coding for rRNA = rDNA = genes for rRNA. NOR is permanent -- is part of DNA. .

b. Nucleolus forms  at location of one or more NOR's. Several NOR's can cluster together to form one nucleolus. Nucleoli are only visible when NOR is transcribed and ribosomes are being assembled.

c. There are multiple copies of genes for rRNA; often many tandem copies per NOR. 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. Multiple polymerases in euk. Differ in sensitivity to drugs, location in nucleus, & what RNA's they make. (See Becker table. 19-1 for details; for reference only.)

1. Types/Names of polymerases:

    a. RNA pol I --> big ribosomal RNAs. 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)

2. How pol II works (and is regulated)  -- how the genes to be transcribed are selected -- will be discussed at length below.

To review nuclear structure, try problem 4-5, parts A & C. Also look at 3-15, Questions A-C, for protein (5).

V. General Chromatin Structure -- Euchromatin & Heterochromatin   

   A. The Issue: 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 regulatory proteins (generally called transcription factors or TFs in eukaryotes) get to the DNA and how do they manage to carry out (& regulate)  transcription with all those proteins stuck to the DNA? To answer these questions, we need to look first at chromatin structure.  

    B. Composition of chromatin = DNA + associated proteins

1. Associated proteins are mostly histones (small, basic; more details below)

2. Associated proteins includes some (nonhistone) regulatory proteins

    C. States of chromatin

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 Basic States of Chromatin (visible in the light microscope)

a. Heterochromatin

(1). Darkly stained, relatively condensed, genetically inactive 

(2). Two kinds of (interphase) heterochromatin

(a). Constitutive heterochromatin -- always heterochromatic (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.

(3). All DNA (chromatin) is heterochromatic during mitosis.

b. Euchromatin

(1). Stains more lightly, less condensed.

(2) Capable of genetic activity (transcription) as vs. heterochromatin. Normal state of most DNA during interphase. (Most interphase DNA is euchromatic, whether it is transcribed or not.) DNA must be euchromatic to be active, but not all euchromatin is active. 

(3). 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.

(c). Use of DNases. States of folding of (eu)chromatin are often distinguished by effects of treatment with various types of DNase. State of (eu)chromatin will determine relative sensitivity of DNA (while still in chromatin) to degradation by various DNases. DNA that is in tighter areas of chromatin will be more protected from degradation. (Some examples of this will be discussed below and are in problem sets.)  

(d). Correlation between folding and function. In general, more active (transcribed) chromatin is looser, but the situation is complex -- see details below.

 VI. Histones & Details of Chromatin Structure

   A. Structure of Chromatin -- how are proteins and DNA arranged? The evidence.

1. Appearance in EM -- low salt ("beads on a string" appearance) vs physiological salt (30 nm fiber). See Becker fig. 16-17 or Purves fig. 9.7 (9.8).

2. Results of treatment with DNase (microccocal nuclease; cuts exposed DNA at random; does not cut at specific base sequences.)

a. Treat chromatin with a little mic. nuclease; then remove protein and isolate the DNA, then run DNA on a gel. Get 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. 16-18 & 16-19.)

b. More treatment with nuclease --> resistant core of 140-145 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.

    B. Model for Chromatin (Beads on string level) -- nucleosomes (See small picture in '91 article (8A) and Becker fig. 16-20 or Purves fig. 9.7). 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.

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 big picture in '97 article (8B) --  and books for detailed pictures. (Becker fig. 16-21 or Purves fig. 9.7 (9.8 or 14.3)) Stages of folding are as follows (details in table):

1. Nucleosomes. DNA + histones --> Chain of nucleosomes; about 1/7 original length of DNA (see table below).

2. 30 nm fiber. Chain of nucleosomes folds back on itself (supercoils) --> 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. (See 97 article.)

3. Loops. 30 nm fiber --> 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. 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 8B.

* Packing ratio = length of DNA or DNA/protein complex relative to original length of DNA.