C2006/F2402 '11 OUTLINE OF LECTURE #9

(c) 2011 Dr. Alice Heicklen & Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/18/2011 09:34 AM .

Handouts:  9A Regulatory Elements & Picture of a typical Eukaryotic Gene (in Word, not pdf)
                   9B Transcription Complex & Modular Regulation -- this handout is posted in Courseworks

For the nucleosome movie shown in class, go to http://www.hhmi.org/biointeractive/dna/DNAi_packaging_vo2.html

I. How Do you turn a Eukaryotic Gene On?

    A. The Problem: Need to unfold/loosen chromatin before transcription is possible. Can't just add RNA polymerase (& basal TFs) to DNA and start transcription. DNA is in chromatin and must be made accessible.

    B. So how can transcription occur?

1. Need multiple steps not found in prokaryotes

a. Must de-condense (loosen up) euchromatin  to a transcribable state = relatively loose (compared to heterochromatin and compared to inactive euchromatin). Pull out 30nm fiber to beads-on-a-string stage?

b. Many transcription factors (TF's) must bind to DNA first -- before RNA polymerase binds.

c. Polymerase must bind to TF's (not directly to the DNA) to get actual transcription.

 2. What changes state of chromatin?  (To tighten or loosen.)

a. Remodeling proteins: these are responsible for moving and/or loosening up nucleosomes. See Sadava fig. 16.19 (14.17).  These may be a separate set of proteins or the TF's that activate the modifying enzymes.

b. Enzymes that modify histone tails. Changes in modification may have a direct effect and/or affect binding of regulatory proteins. Some examples:

(1). Phosphorylation of H1 occurs in M; changes in kinase and phosphatase activity affect state of histones and folding of chromatin in parallel with changes in lamins as discussed last time.

(2). Acetylation of lys side chains of histones. Acetylation of histones → more active, looser chromatin. Acetylation of H3 & H4 is higher in active chromatin.

(3). Methylation -- Effects depend which amino acid side chains in which position of the protein are methylated -- some modifications increase likelihood of transcription, and some decrease it. (DNA can be methylated too; see below.)

c. Methylation  of DNA -- In most organisms, both DNA and histones can be methylated. (Methyl groups can be added to C's in DNA as well as side chains of AA.) 

• Usually, but not always, DNA methylation is higher in more inactive/condensed chromatin.

• In some organisms, there is no methylation of DNA.

d. Overall histone modification 'code' -- it is possible that each combination of modifications to the histone tails has a specific meaning. For a full explanation (fyi) see Alberts. (Or go to PubMed at http://www.ncbi.nlm.nih.gov/sites/entrez , click on books on upper right, and enter 'histone code' in the search term box. )

3. What triggers the tightening or loosening process? Do TF's come first or remodeling/modification enzymes? Current Model

a. Regulatory TF's (activators) bind first -- that triggers remodeling, modification, etc. Loosens up the chromatin in the area to be transcribed.

b. Basal TF's bind later -- After chromatin is loosened up, basal TF's (& possibly more regulatory TF's) can bind to the DNA, pol II can bind to the TF's, and transcription occurs.

4. How does this fit with the DNase sensitivity results?

a. Loosest -- Regions where transcription factors bind -- have nucleosomes removed &/or very loosened up = hypersensitive sites.

b. Looser -- Regions being transcribed -- have nucleosomes somehow "loosened up" or "remodeled" but not removed.

c. Loose -- Regions not being transcribed -- have regular nucleosomes ('loose', relative to heterochromatin, but 'tight' or 'not so loose' compared to transcribed euchromatin.) Regions that are not transcribed are often in euchromatin, not in heterochromatin.

II. Details of transcription in eukaryotes (as vs. prokaryotes) See Becker Ch 21, pp 660-664 (665-670).

    A. More of everything needed for transcription in eukaryotes.

1. Multiple RNA Polymerases (see last lecture). We will focus on pol II (makes mRNA).

2. More proteins -- Need TF's, not just RNA pol.

3. More Regulatory Sequences -- many dif. ones bind dif. TF's

4. An Overview & Some terminology

a. Control elements/sequences -- cis vs trans acting. (See 2nd table on handout 9A.)

• Cis-acting regulatory element =  affects only the nucleic acid molecule on which it occurs. Usually is a DNA sequence that binds some regulatory protein.

• Trans-acting regulatory element = affects target nucleic sequences anywhere in the cell. The regulatory sequence codes for a regulatory molecule -- usually a protein -- that binds to a target -- usually a DNA sequence.

• The term "trans acting" can be used to refer to the regulatory molecule (usually a protein) or to the DNA sequence that codes for it.

b. How Trans-acting and Cis-acting elements work together

• Cis acting elements = DNA itself = same in all cells of multicellular organism = target of trans acting regulatory molecules.

• Trans acting regulatory molecules = product of DNA = TF's & other molecules = different in different cell types and at different times.

• In euk. the number of different types of cis and trans acting control elements is much  larger than in prokaryotes. What are they like? See below.

c. Regulatory Proteins -- Positive vs Negative Control. 

• Regulation can be "+" or "-" depending on the function of the protein

• Negative control --  If regulatory protein blocks transcription.

• Positive control -- If regulatory protein enhances transcription.

•  Euk vs. Prok. -- Negative control (use of repressors) seems to be more common in prok.; positive control (use of activators) more common in euk. 

• How you tell positive and negative control apart -- by effects of deletions.

    B. Details of regulatory (cis acting) sites in the DNA. Prokaryotes have promoters and operators. What sequences do eukaryotes have in the DNA that affect transcription? (The following discussion refers mostly to regulation of transcription by RNA pol II. See texts esp. Becker for details about promoters etc. for pol I & III.) See Sadava Fig. 16.15 (14.14) or Becker fig.23-21 or handout 9A for structure of regulatory sites for a typical protein coding gene. Three types of regulatory sites:

1. Core Promoter

a. Numbering. Position of bases is usually counted along the sense strand from the start of transcription.

(1). "Start" = Point where transcription actually begins (usually marked with bent arrow) = zero.

(2). Upstream and Downstream

    (a). Downstream = Going toward the 3' end on sense strand = in direction of transcription)

    (b). Upstream = Going toward 5' end on sense strand = in opposite direction from transcription.

(3). Numbering -- some examples

    (a). +10 = 10 bases downstream from start = 10 bases after start of transcription.

    (b). -25 = 25 bases upstream  from start = 25 bases before reaching start of transcription.

    (c). +1 = first base in transcript; one that gets a cap (modified base attached to 5' end).

(4). Numbering -- misc. features

     (a). There is no 'zero' base, just as there is no 'zero' year between BC and AD and no zero hour between am and pm.

     (b). In some cases, the position of bases is counted along the sense strand from the start of translation. If it is done this way, the A in the first AUG is +1. However, numbering is assumed to be from the start of transcription unless specified otherwise.

    (c). TF's, RNA pol, etc. bind to grooves in double stranded DNA, not to one strand. However, positions in the DNA are usually specified in terms of the sense strand only. This does NOT mean that the protein binds only to the sense strand.

b. Core Promoter Itself Core promoter is defined by what you need to allow RNA polymerase to start in the right spot. What is included in it?

(1). Actual point for start of transcription (where bent arrow is)  plus a few bases on either side of 'start.' Usually includes a few bases of the 5' UTR (untranslated region). 

(2). Binding sites: Part where basal TF's and RNA polymerase binding starts -- usually section just upstream (before) start point. Often includes short sequence called a TATA box (usually about 25 bases before start point). 

(3). Additional Features:  Often includes some additional or different sequences besides those specified. Not all promoters of Pol II are the same. (If you are interested in details, see Becker 21-12b (13 b), or 23-21)

2. Proximal Control Elements.  (Proximal = Near).

a. Location: Near core promoter and start of transcription; usually "upstream" (on 5' side of start of transcription.) Usually includes regulator elements up to -100 or -200 (bases). 

b. Terminology: Sometimes considered part of core promoter.

c. Function: Binding of appropriate proteins promotes or inhibits transcription. Identified by effects of deletions. Sequence and mechanism of action varies. 

3. Distal Control Elements (Distal = Far)

a. Two kinds: Enhancers & silencers. These control elements can decrease (silencers) or increase transcription (enhancers).

b. These can be quite far from the gene they control (in either 5' or 3' direction = upstream or downstream). Can be in introns or in untranscribed regions. 

c. These can work in both orientations -- Inverting them has no effect, unlike with promoters. See Becker fig. 23-22 (or handout 9B).

d. Mechanism of action -- bind TF's; see below.

4. Terminology & Misc. Details -- this is for reference; may not be discussed in class.

a. Boxes = short sequences that are found in regulatory regions (ex: TATA box)

b. Consensus sequences = sequence containing the most common base found at each position for all sequences of that type. Any individual version of sequence is likely to be different from the consensus at one or more positions. (Ex: TATAAAA = consensus sequence for TATA box. Means T is most common base in first position, A is most common in second position, etc.)

c.  For multicellular organisms, term "operator" is not used for site/DNA sequence where a regulatory protein sits. Why? Because no polycistronic mRNA & no operons in higher eukaryotes. (Are some in unicellular euk.)

    C. How do Basal Transcription Factors work?

    D. How do Regulatory or Tissue Specific TF's  Work?

1.  Different ones are used in different cell types or at certain times. Not all are needed in all cells. See Becker fig. 23-24.

2. Properties

a. Bind to areas outside the core promoter -- usually to enhancers or silencers (distal control elements) but sometimes to proximal control elements

b. When regulatory TF's bind, can decrease or promote transcription.

(1). Activators. TF's called activators if bind to enhancers and/or increase transcription.

(2). Repressors. TF's called repressors if bind to silencers and/or decrease transcription.

c. How regulatory TF's affect transcription: DNA thought to loop around so silencer/enhancer is close to core promoter.  TF's on enhancer help stabilize (or block) binding of  basal TF's directly or indirectly to core promoter. (See Becker fig. 23-23 or Sadava fig. 16.15 (14.14) and section on regulatory TF's below.)

d. Euk. vs Prok. repressors -- both 'repressors' interfere with transcription, but mechanism of action is different.

e. Role of Co-activators -- Proteins that bind to TF's on the enhancer and influence transcription (but don't bind directly to the DNA) are often called co-activator (or co-repressor) proteins. There are 2 ways co-activators affect transcription:

(1). Act as mediator -- Connect two parts of the transcription machine.  One part of mediator binds to TF (which is bound to enhancer or silencer) and other part of mediator binds to basal transcription factors (or pol II) on core promoter and/or proximal control elements. Mediator = usual name of complex of co-activators that act this way.

(2). Modify state of chromatin. Bind to TF on enhancer and loosen up chromatin in gene to be transcribed. Remodeling proteins and histone modifying enzymes are included in this category.

To review gene structure & TF's, try problems 4R-2, 4R-5A & 4R6-A.

f. Co-ordinate control.  A group of genes can all be turned on or off at once in response to the same signal (heat shock, hormone, etc.).

(1). Prokaryotes vs. Eukaryotes: Both prok. and euk. exhibit co-ordinate control, but mechanism is different. (See table below.)

(2). Location of coordinately controlled genes

(a). In prokaryotes, coordinately controlled genes are located together in operons.

(b). In eukaryotes, coordinately controlled genes do not need to be near each other -- they just have to have the same (cis acting) control elements. See Sadava fig. 16.17 (14.16). 

(3). Control elements:

(a). All genes turned on in the same cell type and/or under the same conditions share the same control elements -- therefore these genes all respond to the same regulatory TF's. Result is multiple mRNA's, all made in response to same signal (s). 

(b). Most genes have multiple (cis acting) control elements. Therefore transcription of most genes is affected by more than one TF.

(c).  Transcription of any particular gene depends on the combinations of TF's, not just one, available in that cell type.

(4). Differences in TF's. Different cell types make different regulatory TF's.   Therefore different groups of coordinately controlled genes are turned on/off. See Becker fig. 23-24.

(5). Comparison of situation in prokaryotes vs multicellular eukaryotes: 

g. Modular Regulation -- Handout 9B, bottom. Different signals and so different
transcription factors bind to different enhancers on the same gene to
turn the gene on in different cells &/or a different times.

III. Overall Regulation of Eukaryotic Gene Expression  --  What has to be done to make more or less of a protein?  A different protein? What steps can be regulated?

    A. If cells make different proteins, how is that controlled? If two eukaryotic cells (from a multicellular organism) make different proteins, what is (usually) different between them?

Examples: Chicken oviduct cells make ovalbumin -- chicken RBC make globin*

                Human liver cells make transferrin -- human precursors to RBC make globin

*Note: chicken RBC, unlike human RBC, have nuclei

1. Is DNA different? (No, except in cells of immune system.)

2. Is mRNA different? (Ans: yes). This means you can get tissue specific sequences from a cDNA library. (cDNA library = collection of all cDNA's from a particular cell type.) DNA from each cell type is the same; mRNA and therefore cDNA is not. See Becker fig. 23-20.

3. Is state of chromatin usually different? (Ans: yes) How is this tested? Method & result described previously. See figure 23-17 in Becker.

To review transcription, if you haven't done them yet, try problems 4R-5 and 4R-6A.

4. Is any step after transcription different? Post-transcriptional & translational regulation will be discussed by Dr. M after development.

Next week:  Development. Dr. H will continue to explore transcriptional differences since transcriptional regulation is a big player in development.