C2006/F2402 '09 OUTLINE OF LECTURE #9

(c) 2009 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/23/2009 05:45 PM

Handouts (not on web): 9A & 9B  (nucleosome & chromatin structure). Similar to Becker figs. 18-20 & 18-21.

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

    A. Pore structure -- hard to explain; will be demonstrated. See Becker fig. 18-27 & 28 and/or Sadava fig. 4.8 (4.9)

    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:

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. Additional proteins (that we will ignore) are required as well. See Becker fig. 18-30 for a model.

*A middle man protein is required to enter or exit the nucleus, but different ones are used in for entry vs exit. Exportin is needed to get out of the nucleus, while importin is needed to get in.

** This is how soluble nuclear proteins are imported into the nucleus  Integral (transmembrane) proteins of the nuclear membrane (nuclear envelope) 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.

3. Transport is bidirectional -- mRNA, ribosomal subunits etc., must go out (see below) and proteins 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. Exit requires an exit signal (NES) and the transporter protein exportin.

To review nuclear transport, try problem 4-2.
 

II. 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 lamina = web supporting the nuclear envelope -- see Becker fig.  18-31(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; may provide attachment sites for chromosomes and nuclear pore complexes.

    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.

• 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 similar, but not identical, in both directions.

• FYI: 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.

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

b. Nucleolus  = structure seen in interphase; not in meiosis/mitosis.

• Nucleolus forms during interphase 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 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)

d. 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. 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 (& regulate)  transcription with all those proteins stuck to the DNA? To answer these questions, we need to look first at chromatin structure.  
 

II. 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

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 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. 

(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.

    E. Modifications of histones -- helps tighten up or loosen chromatin

To review nucleosome structure, try problems 4-1, 4-3 A, 4-6 C, 4-9 A, 4-7 B, & 4-10 A.

IV. Does Genetic Activity Correlate with States of Euk. Chromatin? How to test?

    A. Polytene chromosomes  -- Special Interesting Case. Will not be covered in lecture; is here FYI.  If you are interested, see Becker 23-15 & 16.

1. Advantages:

a. Special case where many chromatids -- about 1000 -- are lined up together with genes and loops of chromatin aligned;  therefore can see differences in state of folding in light microscope.

b. Can compare changes in folding of chromatin and level of transcription (by incorporation of labeled U) in a single tissue as it responds to hormones that alter gene expression.

2. Disadvantage: Can't compare regions of active/inactive chromatin from many dif. tissues. (Polytene chromosomes occur only in a few tissues of selected organisms.)

3. Results: Active (transcribed) regions are clearly looser -- individual loops stretched out more. (See Becker  Ch. 23)

    B. Results of treatment with DNase I (of ordinary chromosomes/chromatin) -- How folded is Euchromatin? Is it all the same?

1. The problem -- Not all euchromatin is transcribed in any one cell -- is it all folded to the same degree or not?

a. All (eu)chromatin looks about the same in the light microscope.  Therefore indirect methods (such as DNase treatment) are necessary to test state of folding.

b. Use of DNases.  States of folding of (eu)chromatin are often distinguished by effects of treatment with various types of DNase. How? 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.)  Therefore,  state of (eu)chromatin can be deduced from relative sensitivity of DNA (while still in chromatin) to degradation by various DNases.

2. Method -- see Becker fig. 23-17. Want to examine chromatin region containing a particular gene, say globin genes, from dif. tissues. (Note: this region is euchromatic in interphase in all these tissues, even though it is not expressed in all of them. No gross difference is visible; that's why indirect methods are necessary.) 

a. What you want to do: Fish out section of chromatin containing globin genes (from cells that make globin and cells that don't) and test state of chromatin folding.

b. What you have to do: Need to test for folding indirectly, so you need to do experiment in reverse. You distinguish states of folding of chromatin first (indirectly) and then find which state contained the genes of interest, say, globin genes.

c. How you do it: Treat chromatin with DNase, differentially degrading DNA in active and inactive chromatin. Then use probe to test for state of  genes of interest and see if the genes were degraded or not. 

d. Details of DNase treatment:

(1). Treat chromatin -- not DNA -- with DNase. Then isolate DNA and see what state it's in.

(2). DNase used is not micrococcal nuclease.

(a). Usually use DNase I, a different enzyme from one used previously to get a "ladder". 

(b). DNase I cuts the DNA differently than micrococcal nuclease. DNA that is simply wound around nucleosomes is not totally protected from DNase I -- further folding up is required. So resistance to DNase I is a measure of how tightly the nucleosomes are folded in on themselves. DNase I does not cut preferentially at any particular sequence or at any particular place relative to the position of the nucleosome. Therefore it does not give a "ladder." 

(3). Can vary conditions -- amount of enzyme, time, etc. to distinguish various degrees of sensitivity to the enzyme. 

(4). Can test chromatin from many different tissues, say erythrocytes (in chickens -- still have nuclei) vs. brain

(5). Can test state of many different genes using diff. probes (in step e below).

e.  Expected result: If chromatin is relatively loose, DNA will be unprotected and readily degraded by DNase I. (There will be nothing left to hybridize to the probe for, say, globin.) If chromatin is relatively tight, DNA in that region will NOT be easily degraded. (And there will be DNA to hybridize to probe.)

f. How to measure state of DNA (after treatment of chromatin with DNase I). Cut purified DNA with restriction enzyme and do a Southern blot with a probe to the gene of interest.  Details:

(1). Prepare DNA. Extract DNA (remove histones); treat naked DNA with restriction enzyme (to give pieces of reasonable size IF DNA was protected from DNase I). If DNA was in loose region of chromatin, it will already have been degraded by DNase I.  

(2). Find regions corresponding to known genes. Do Southern: Run restriction fragments on gel, blot, hybridize to labeled probe to identify DNA from regions of interest (say, globin genes). Only DNA from areas with relatively tight chromatin will give clear, undegraded (labeled) bands on the gel.

To review how this is done, try problem 4-9.

3. Results of treatment of euchromatin with DNase I and other nucleases.

a. Almost all eukaryotic DNA is associated with some protein. How do we know? -- Almost all DNA is much more resistant to digestion by DNase I than naked DNA

b. Almost all eukaryotic DNA is in nucleosomes. All DNA but the very loosest euchromatin (see d) is in nucleosomes, and gives a ladder when treated with microccal nuclease. 

c. Actually transcribed (coding) DNA is more sensitive to DNase I than ordinary euchromatic DNA. But transcribed DNA is much more resistant than naked DNA -- it is still in nucleosomes. 

d. Hypersensitive sites exist -- these are the only sections of euchromatin that are not in nucleosomes. See Becker fig. 23-18 (21-18).

(1). Some hypersensitive sites found = very sensitive regions (100X more sensitive to degradation by DNase I than heterochromatin; 10X more sensitive than active euchromatin.) Degraded by very low amounts of DNase I.

(2). Hypersensitive sites correspond to sites without nucleosomes 

(3). DNA in hypersensitive sites is not naked -- it has other proteins, but not histones.

(4). Hypersensitive sites correspond to regulatory, not coding, regions (in areas of active transcription).

(5). Hypersensitive sites are different in different tissues -- different genes are 'turned on' (transcribed) in each cell type. DNA is the same in (almost) all cells, but only the genes that are 'turned on' (in that cell type) will have hypersensitive sites.

(6). Why are hypersensitive sites so sensitive to DNase? Transcription factors (regulatory proteins) have replaced histones and the other proteins don't protect the DNA as well as histones do.

    C. Possible States of Interphase Chromatin -- what is inferred from all the results, and the implications. (See table on handout. )

a. Super Tight  = heterochromatin = essentially not fully uncoiled from mitosis. Genetically inactive.

b. Euchromatin -- 3 major states?

(1). Loose. Looser than heterochromatin, but not being transcribed (genetically inactive) at the moment or recently. 30 nm fiber? 

(2). Looser -- but still has nucleosomes. 10 nm fiber or beads-on-a-string state. Stretched out loops? Example: coding region that are active -- they are actually being transcribed. (Also includes genes that were transcribed recently, or are next to transcribed regions, etc.)

(3). Loosest or hypersensitive region (missing some histones; has regulatory proteins instead). Example: active promotor (Promotor = region around where transcription starts; it's where RNA polymerase and associated proteins bind. More on this next time.) 

c. A caution: There are probably intermediate states, and the ones above are simply the only ones that have been clearly distinguished using the methods currently available. For example, all inactive euchromatin is probably not the same. 

To review the differences in states of chromatin, uses of DNase, etc., try the rest of 4-3, 4-9 & 4-10.