BMB 251 1st Edition Lecture 25 Outline of Last Lecture I. Coding/Template strand and 5’, 3’ endsII. Promoter sequences III. snRNPsIV. SpliceosomeOutline of Current Lecture V. Cell differentiationVI. Types of cellular controlVII. Gene Regulatory ProteinsVIII. Negative controlIX. Positive controlCurrent Lecture- Clicker Question 1: Consider the cartoon of the sequence specific DNA binding factor. Not the two “palindromic” “half-sites” separated by 5 bases what can you surmise about the structure ofthis complex?5’ TAACACCGTGCGTGTTG3’3’ATTGTGGCACGCACAAC5’o The DNA binding protein binds as a dimer They will create two mirror image major grooves, which bind with each other to create a dimer- Cell differentiation depends on gene control/expression, not changes in the actual nucleotide sequenceo Cell types in multicellular organisms come from synthesis of different RNA and proteins (structural proteins of chromosome, DNA/RNA polymerase, ribosomal proteins, etc.)- Some proteins can only be detected in their specialized cell (ex. Hemoglobin in red blood cells)- Most specialized cells are capable of altering their patterns of gene response due to extracellularcues- **Different cell types often respond differently to the same extracellular signal (increase protein production, decrease production, not react at all to signal, etc.)These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- There are many regulations where gene expression can be controlled from the process of creating a protein from a geneo Transcriptional control (most prominent): controls when/ how often a gene is transcribedo RNA processing control: controls splicing and processing of RNA transcriptso RNA transport and localization control: selecting which mRNA is exported out of nucleus and where in the cytosol it will be localizedo Translational control: selecting which mRNA in cytosol are translated by ribosomeo mRNA degradation control: selectively destabilizing certain mRNA o Protein activity control: selectively activating, inactivating, degrading or locating specific proteins after they have been synthesized- Gene regulatory proteins: turn genes on or off by binding noncovalently to the major and minor grooves of DNA- The outside of DNA double helix contains DNA sequence info that gene regulation proteins can recognize without having to open the double helixo Gene regulatory proteins generally make contact with major groove of double helix because this is the only section that shows distinct differences between the four DNA bases (H-bond donors, H-bond acceptors, hydrophobic patches, etc.)- Gene regulatory protein recognizes a specific DNA sequences because the surface of the protein is extensively complementary to the special surface features of the double helix in that regiono Creates H-bonds, ionic bond and hydrophobic interactionso Each individual contact is weak, but when they are all added together, it cerates highly specific and strong protein-DNA interaction- Many Gene regulatory proteins contain one or another of a small set of DNA-binding structural motifs (allow DNA and protein to fit together perfectly)o Helix-Turn-Helix: constructed from two alpha helices connected by a short, extended chain of amino acids (create “turn”) Recognition helix: the more C-terminal helix, which fits into the major groove of DNA Part of polypeptide outside of H-T-H domain also makes important contacts withDNA, helping to “fine-tune” the interaction N-terminal alpha helix functions primarily as a structural component that helps to position the recognition helix Many bind as symmetric dimers to DNA sequences composed of two very similar“half-sites” (two copies of recognition helix separated by exactly one turn of DNA helix) Composed solely of amino acidso Zinc fingers: includes one or more zinc atoms as its structural components Two types: 1. Occur in proteins which activate rRNA synthesis: zinc holds alpha helix and beta sheet together- Usually found in clusters where each has their alpha helix interacting and forming an almost continuous stretch along the major groove- Strong and specific DNA-protein interaction is built up through repeatingbasic structural units 2. Occur in large family of intracellular receptor proteins: two alpha helices are packed together with zinc atoms - Usually form dimers that allow one of the two alpha helices of each subunit to interact with major groove of DNA **Both use zinc as structural component and alpha helix to interact with the major groove of DNAo Beta sheets: two stranded beta sheet, with amino acid side chains extending from sheet toward DNA, reads info on major groove (instead of alpha helix seen in previous motifs)o Few DNA-binding proteins use peptide loops to read nucleotide sequences, instead of alpha helices and beta sheets (Ex. P53 recognizes nucleotide pairs from both major and minor groove using these loops)o Leucine zipper motif: two alpha helices (one from each monomer) are joined together toform a short coiled-coil  combines portion of proteins responsible for dimerization andDNA binding Helices held together by hydrophobic interactions of amino side chains (usually on leucines) which extend from one side of each helix  Creates Y-shaped structure of alpha helices, which allow their side chains to interact with the major groove Heterodimerization: example of combinatorial control, in which combinations of different proteins, rather than individual proteins, control a cell process o Helix-Loop-Helix motif: short alpha helix connected by a loop to a second, larger alpha helix Flexibility of loop allows one helix to fold back and pack against the other- DNA molecule is highly negatively charged and therefore moves rapidly towards the positive electrode in an electrical field- In polyacrylamide-gel electrophoresis, DNA molecules are separated by size o Smaller DNA molecules penetrate gel easiero Larger proteins bound to DNA increase the retardation of DNA movemento **Gel mobility shift assay: allows trace amounts of sequences-specific DNA-binding protein to be detected via radioactive labels- Genes can be switched on and off- Negative control: when a protein (repressor) binds to an operator (sequence within a promoter) and blocks access of RNA polymerase and therefore RNA transcription- Positive