CSB349 Lecture 6 Notes

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Cell and Systems Biology
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Alan Moses

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CSB349 Lecture 6 – Transcription II: Promoters and sequence-specific DNA binding proteins Slide 2 – Regulating gene expression: The role of promoters - The promoter is upstream of the transcription start - There are two major parts of the promoter o Core promoter  Contains DNA recognition elements for the basal transcriptional machinery that is going to be involved in transcription start o Proximal promoter  A collection of regulatory elements that are important for gene regulation  All kinds of different elements can appear in the proximal promoter region  This is where regulatory sequences are found - The regulatory elements in the core promoter, and usually the proximal promoter, is that the regulatory elements are location-dependent o The core promoter needs to be right near the transcription start site so that it is efficient o The position and orientation of the promoter matters Slide 3 – Experimental characterization of functional promoters - There is no well understood language or code of what makes the promoter work - We don’t know what the important elements are in the promoter - The classical experiment for studying promoters is called promoter bashing o Break the promoter into pieces o Figure out which ones work and which ones don’t, which will tell us where the functional sequences are - Steps o Take a chunk of the promoter o Chop up the promoter into different pieces o Clone them into a series of reporter constructs  The plasmid has a reporter gene, instead of the normal gene o Put the reporter construct into a cell where you can measure the concentration of the reporter gene  There are different ways to measure the reporter gene - Results o Pieces 1 and 2 contain all of the necessary information for full gene expression o Pieces 3 and 4 contain some minimal information for some gene expression o Piece 5 does not contain any information for gene expression - Interpretation o The core promoter is on pieces 1, 2, 3, and 4 maybe o There are proximal promoter information on pieces 1 and 2 o The core promoter is absolutely required for any gene expression o The proximal promoter is giving some extra expression Slide 4 – Experimental characterization of functional promoters - Another kind of promoter bashing is to systematically delete pieces of the promoter to look for effects on gene expression - You sequentially knockout pieces of the promoter Slide 5 – Identifying protein binding sites in DNA sequences (in vitro) - To figure out where different proteins might be binding to the promoter - This technique is called DNase I footprinting assay - DNase I digests DNA when the DNA is open - To figure out which parts of DNA are bound by protein - The functional elements inside promoters are the binding sites of DNA binding proteins - Steps o Make a whole bunch of fragments that are identical copies of the same promoter o Label one end of the promoter fragment (e.g., radioactive label) o Add nuclear extract (purified proteins) to the fragments  The proteins are binding to a specific sequence in the promoter  All the proteins will bind to the promoter in the same place  DNase I digestion cuts the fragments, except where the protein is  Wash the DNase I  Run on the same gel o Compare what you see when you don’t add the nuclear extracts  Treat with a limited amount of DNase I  None of these are bound by proteins  The DNase I can cut each fragment about once, on average  Run on a gel, which will have almost all sizes of fragments - Results o See fragments that are missing o There were no fragments that were cut at that length o The protein was blocking the DNase I from cutting at those lengths o The missing fragments are called the footprint o The protein has left a footprint on the DNA o You can infer that something was binding there to the promoter Slide 6 – Promoter regulatory sequences - There are specific regulatory sequences that those DNA binding proteins are recognizing o Called consensus sequences o How do I figure out the consensus sequence? Slide 7 – Experimental characterization of functional promoters - What is it about the sequence that makes the protein want to bind here? - Go through the sequence and start mutation each possible letter to see whether that disrupts transcription or not o The possibilities are endless o The number of constructs that you need to make are endless - To define the consensus sequence Slide 8 – Defining consensus sequences for regulatory elements - There are faster things you can do to characterize the protein’s DNA binding specificity - To figure out the consensus sequence for the protein - Look through the literature to count the different consensus sequences Slide 9 – Promoter sequences - These are three of the most typical and abundant promoter elements o TATA box  Not the most important of these core promoter elements o Inr element  More common  Around 10 bp around the transcription start site o DPE element  Downstream promoter element  Binding site that is found downstream of the TSS - None of these elements are found in all promoters (e.g., not 100%) o Each promoter has different elements that are responsible for transcription start Slide 10 – Organization of regulatory DNA in multicellular eukaryotes is relatively complex - Enhancers (or cis-regulatory modules) can be located anywhere (e.g., downstream, upstream, intron region) - The organization of regulatory sequences is different between different eukaryotes - The organization of the regulatory information is much simpler in yeast (S. cerevisiae) o Much smaller genome o Few introns o Contain core promoter nearby the TSS o Do not tend to have many enhancers spread throughout o Enhancers and proximal promoter sequences are lumped together o No clear distinction between proximal promoters and enhancers Slide 11 – Sequence-specific transcription factors - The core promoter elements are typically bound by the basal transcription factors (e.g., TATA binding protein, TFIID) - The proximal promoter sequences and the enhancers are typically bound by sequence specific transcription factors o Sequence specific transcription factors are the most important control of gene expression - The sequence specific transcription factors form a large class of proteins and they can be categorized into different families o They can be categorized into different families because they contain DNA binding domains o The organization of transcription factors is modular  They have a specific part of the protein that is responsible for binding to the DNA  There are other parts of the protein that are responsible for interacting with the transcription machinery, HDAC, or HAT that lead to changes in the chromatin or gene expression - They can also contain protein-protein interaction domains o These transcription factors typically do not work as monomers o They will typically bind to other DNA binding proteins o The typical thing is dimerization Slide 12 – Modular structure of transcription factors - These are four random transcription factors - These different transcription factors have different organizations of DNA binding domains, activation domains, and flexible domains - Sometimes they can have two DNA-binding domains - You can typically chop up these different domains and the individual domains will still work o They seem to be independent functional modules Slide 13 – Methods of determining protein-DNA interaction - I told you about one way of figuring out that DNA binding proteins are binding to your promoter - This is another technique that accomplishes something similar, but in this case, you can figure out which DNA binding proteins are binding to your promoter o EMSA (gel shift assay) = in vitro assay  Take the radioactive DNA fragments and run them on the gel without chopping them up, and you will see one band  You are going to put on some kind of cell extract or some kind of purified protein, and you ask “does it change the mobility of the DNA on the gel?”  If proteins are binding to the DNA, it is going to change the mobility pattern of the DNA on the gel  The protein causes a shift in the mobility of the DNA running on the gel - To figure out which sequences are actually bound by a DNA binding protein o Given a large random number of DNA sequences, you can figure out which ones your protein prefers to bind to o Another way of getting the consensus sequence for the protein, in vitro o SELEX = in vitro assay  Make a big library of random DNA sequences  Take the DNA binding protein and FISH out the ones that your protein likes to bind to out of the random pool of dsDNA  You can do this with a gel shift assay  Run the random pool of DNA on the gel  Putting the DNA binding protein might slow down a fraction of the pool  The part that is slowed down is presumably the part that is sticking to your DNA
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