(c) 2014 Dr. Alice Heicklen & Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/26/ :57 PM

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1 C2006/F2402 '14 OUTLINE OF LECTURE #11 (c) 2014 Dr. Alice Heicklen & Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/26/ :57 PM Handouts: 10C -- Typical Eukaryotic Gene, and Cis & Trans 11A -- Regulatory Regions, Combinatorial Control & Co-ordinate Control.(posted on CW for registered students) 11B -- Transcriptional Regulation, Crystallin & Modular Promoters (posted on CW for registered students) You may also need to refer to 10C. Reminder: paper copies of all handouts are handed out in class and available after class on the 7th floor of Mudd. For the nucleosome/histone movie go to 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 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 loosened up (compared to heterochromatin and compared to inactive euchromatin). Pull out 30nm fiber to beads-on-a-string stage? (1). Loosest -- Regions where transcription factors bind -- have nucleosomes removed &/or very loosened up = hypersensitive sites. (2). Looser -- Regions being transcribed -- have nucleosomes somehow "loosened up" or "remodeled" but not removed. (3). 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. 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 2. What changes state of chromatin? (To tighten or loosen.) Binding of regulatory proteins, modification of histone tails, and methylation of DNA bases, are all known to occur. II. Details of transcription in eukaryotes (as vs. prokaryotes) See Becker Ch 21, pp ( ). A. More of everything needed for transcription in eukaryotes. 1. Multiple RNA Polymerases (see end of lecture 7). We will focus on pol II (makes mrna).

2 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 10C.) 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. (See 1st table on handout 11A.) Regulation can be "+" or "-" depending on the function of the protein Negative control -- If regulatory protein blocks Positive control -- If regulatory protein enhances 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? Note: 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) or Becker fig or handout 10C for structure of regulatory sites for a typical protein coding gene (transcribed by pol II). Two types of regulatory regions: 1. Basal Promoter a. Numbering. Position of bases is usually counted along the sense strand from the start of

3 (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 (3). Numbering -- some examples (a). +10 = 10 bases downstream from start = 10 bases after start of (b). -25 = 25 bases upstream from start = 25 bases before reaching start of (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. Basal Promoter Itself -- Basal (or 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. Regulatory Regions = all regulatory regions besides the basal promoter a. Terminology. Also called control elements. Becker (& some problems in the problem book) distinguish between proximal control elements (near to basal promoter) and distal ones (far from basal promoter). However, most scientists don't differentiate.

4 b. Mechanism of action -- bind TF's and increase or decrease transcription; see below. c. Enhancers vs silencers. These control elements can decrease or increase transcription, depending on what TFs bind to them. These control elements are usually named after their primary effect on (1). Enhancers -- primary effect is to increase transcription (2). Silencers -- primary effect is to decrease transcription. d. Regulatory regions 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. e. These can work in both orientations -- Inverting them has no effect, unlike with basal promoters. See Becker fig f. Discovery: Identified by effects of deletions. 3. Terminology & Misc. Details -- this is for reference; may not be discussed in class. a. Boxes = 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? 1. Same in all cells. Needed to start transcription in all cells. See Sadava fig (16.14) or Becker fig (21-14). 2. Properties a. Many basal TF's needed. b. Basal TF's for RNA pol. II. (1). Terminology: Basal TF's for pol II are called TFIIA, TFIIB, etc. (2). Major one is TFIID; it itself has many subunits. Most studied subunit is TBP (TATA binding protein -- See Becker fig (21-15).) Recognizes TATA box when there is one. (3). Other polymerases have TF's too, but TF's for pol II are of major interest, since pol II mrna c. Basal TF's bind first to core promoter, and then RNA pol binds to them. Takes a lot of proteins to get started. RNA polymerase does not bind directly to the DNA.

5 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 Properties a. Bind to areas outside the basal promoter -- to enhancers or silencers b. When regulatory TF's bind, can decrease or promote (1). Activators. TF's called activators if bind to enhancers and increase (2). Repressors. TF's called repressors if bind to silencers and decrease 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 or Sadava fig (16.15) and section on regulatory TF's below.) See Handout 11A. 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 corepressor) 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 coactivators that act this way. (See Handout 11A.) (2). Modify state of chromatin. Bind to TF on enhancer and loosen up chromatin in gene to be transcribed. Histone modifying enzymes & remodelers are included in this category. To review gene structure & TF's, try problems 4R-2, 4R-5A & 4R6-A. 3. Types of control by TFs a. Combinatorial control: TFs work in combination. See Handouts 11A & 11B. (1). Most genes have multiple (cis acting) binding sites for TFs. Therefore transcription of most genes is affected by a combination of TFs, not just one TF. (2). What genes are transcribed? Transcription of any particular gene depends on the combination of TFs available in that cell type. (Handout 11B.) (3). How many TFs? A different TF is not required to turn on each gene -- For example, 4 TFs can turn on more than 10 genes b. 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.). See Handout 11A.

6 (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). (3). Regulatory regions = Control elements: (a). All genes turned on in the same cell type and/or under the same conditions share the same regulatory regions -- 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). Transcription of any particular set of coordinately controlled genes depends on the combination 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 (5). Comparison of situation in prokaryotes vs multicellular eukaryotes: Prokaryotes Multicellular Eukaryotes Coordinately controlled genes are Linked Unlinked Messenger RNA is Polycistronic (1 mrna/operon) Moncistronic (1 mrna/gene) Operons? Yes No Control elements are found Once per operon Once per gene Control can be positive or negative but is more often Negative -- repressors needed to turn gene off Positive -- activators needed to turn gene on. c. Modular Regulation -- Handout 11B. Different combinations of transcription factors can turn on the same gene (or group of genes) in different cells &/or at different times. (1). Role of enhancer: Different enhancers respond to different signals (2). More than 1 regulatory region per gene (3 million hypersensitive sites in humans) Transcription of the average gene is affected by different regulatory regions. 95% of regulatory regions are located 2.5 KB or more away from transcription start sites. (3). Multiple TFs bind each regulatory region

7 (4). Different TFs in different cells can turn on the same gene 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 Is state of chromatin usually different? (Ans: yes) How is this tested? One method -- by digestion with DNase. See handout 10-A and figure in Becker. To review transcription, if you haven't done them yet, try problems 4R-5 and 4R-6A. 4. Why is the mrna different? a. Transcription is different. Only selected genes are transcribed in each cell type, and RNA's from those genes are processed to make mrna. (For an experiment that shows this, see figure (23-19) in Becker.) b. Is the difference due entirely to differences in transcription? Splicing and processing of same primary transcripts can be different (in different cells or at different times). Different mrna's (& therefore proteins) can be produced from the same transcript by alternative splicing and/or poly A addition. More details next time. B. How can amount of protein synthesized be controlled? If cell makes more or less protein, which step(s) are regulated? Transcription is main point of control, but other steps are often regulated too in eukaryotes. How this works, and comparison to the situation in prokaryotes, next time. Next time: Details of Post Transcriptional & Post Translational Regulation