Cell And Molecular Biology Lecture 9 Control Of Gene Expression PDF

Cell and Molecular Biology Lecture 9: CONTROL of GENE EXPRESSION From CHAPTER 7 Molecular Biology of the Cell, 4th Ed. Alberts et al. Daniele Provenzano, Ph.D. Virtually every cell of a multicellular organism contai...

Cell and Molecular Biology Lecture 9: CONTROL of GENE EXPRESSION From CHAPTER 7 Molecular Biology of the Cell, 4th Ed. Alberts et al. Daniele Provenzano, Ph.D. Virtually every cell of a multicellular organism contains the DNA encoding all the genes required to reconstruct the entire organism. NEURONS and LYMPHOCYTES from the same organism have very different functions, very different appearance and size. Although both cells contain the same genome they express different genes, they produce different mRNAs and proteins. The cloning of whole organisms demonstrates that each cell contains all the genetic information to reconstruct the entire creature:  Each cell has very specific tasks within an organism and activates only a particular set of the genes at any given time. That is why cells exist that have such different function and appearance all within the same organism. Furthermore, cells can change the expression of their genes (and their appearance) in response to external signals A CELL CAN CHANGE EXPRESSION OF GENES IN RESPONSE TO EXTERNAL SIGNALS: FOR EXAMPLE, DURING STARVATION OR INTENSE EXERCISE, GLUCOCORTICOIDS ARE RELEASED IN THE BODY CAUSING THE LIVER TO CONVERT AMINO ACIDS INTO GLUCOSE. THIS LEADS TO AN INCREASE IN EXPRESSION OF THE GENE ENCODING TYROSINE AMINOTRANSFERASE WHICH HELPS CONVERTING THE AMINO ACID TYROSINE TO GLUCOSE. WHEN THE GLUCOCORTISOID LEVELS IN THE BODY DECREASE, EXPRESSION OF THE TYROSINE AMINOTRANSFERASE GENE DROPS BACK. CONTROL OVER GENE EXPRESSION MUST BE VERY TIGHTLY REGULATED AND VERY PRECISE FOR CELLS TO BE SO DIFFERENT AND RESPOND TO A VARIETY OF DISTINCT SIGNALS.  GENERALIZATIONS: 1. Many processes are common to all cells (in an organism, and in many cases across species) For example, structural proteins of chromosomes, DNA and RNA polymerases, DNA repair enzymes, cytoskeletal proteins, enzymatic reactions central to metabolism, and others… 2. Some proteins are abundant in the specialized cells in which they function and cannot be detected anywhere else. Keratin in surface epithelial cells, hemoglobin in erythrocytes, muscle fibers in muscle cells, etc… 3. Studies of global mRNA content have shown that of the approximately 25’000 genes, each human cells expresses anywhere between 10’000 and 20’000 genes. The expressed genes vary from cell type to cell type. 4. Gene expression can be modulated at different stages of protein synthesis, at transcription, RNA processing, RNA transport and localization, RNA degradation, translation, post translation, and even beyond that stage using mechanisms that mediate protein activity, secretion, and export. SIX STEPS WHERE EUKARYOTIC GENE EXPRESSION CAN BE MODULATED: Critical step HOW DOES A CELL KNOW WHICH ONE OF THOUSANDS OF GENES TO TRANSCRIBE ? 1. Gene regulatory proteins are capable of reading the DNA sequence of the coiled double helix without opening it up: 2. Binding of DNA by proteins causes a sequence-specific change in the shape of the helix:  HOW DO GENE REGULATORY PROTEINS READ THE COILED DNA DOUBLE HELIX ? Each nucleotide confers a unique geometry to the helical DNA structure that gene regulatory proteins are capable of distinguishing:  GENE REGULATORY PROTEINS STRUCTURAL MOTIFS that can read DNA sequences: Gene regulatory proteins can make extensive contact with the DNA through surfaces of the protein that are complementary (good fits) with the double helix in the particular sequence they are intended to recognize. These interactions, although non covalent, are sufficiently strong, and highly specific to the target sequence: DNA-binding motifs usually bind to the major groove of the double helix:  The HELIX-TURN-HELIX (HTH) is one of the most common DNA-binding motifs: The N’- HELIX functions primarily as a structural component that helps to position the recognition helix at the right angle. The C’-RECOGNITION HELIX fits in the major groove of the DNA and is made up of specific amino acids that recognize the DNA sequence. The HELIX-TURN-HELIX (HTH) motif is found in DNA-binding proteins across all species: Most, if not all DNA-binding proteins do so as dimers (in pairs). The two copies of the DNA-recognition motifs are are all spaced exactly by 1 turn of the DNA helix (3.4 nm).  THE NUCLEOTIDES RECOGNIZED BY THE DNA- BINDING PROTEIN LAMBDA CRO ARE SHOWN IN GREEN – NOTE THE SAME SEQUENCE ON BOTH STRANDS IS RECOGNIZED BY THE SAME PORTION OF THE PROTEIN. What is this sequence recognized by Lambda Cro called? Helix-turn-helix (HTH) motifs in eukaryotes are part of a larger structure called HOMEODOMAINS: Homeodomains are composed of three α- helices packed tightly together by hydrophobic interactions. Helix 2 and 3 form a HTH motif, where H3 acts as the recognition helix.  Another family of DNA-binding motifs is called ZINC FINGER In contrast to HTH motifs made up exclusively of amino acids, zinc fingers also use zinc atoms. Zinc fingers are made up of a β-sheet and an α -helix held together by a zinc atom. Zinc finger, like HTH motifs usually bind DNA as dimers. There are several types of zinc finger motifs, they all share the presence of a Zn atom. SHOWN IS THE SAME ZINC FINGER MOTIF IN TWO DIFFERENT REPRESENTATIONS The α-helix of the zinc finger interacts with DNA In this example of a gene-regulatory protein of mice, three zinc fingers bind the target DNA sequence as shown:  Several additional DNA-binding motifs exist: All DNA-binding domains recognize specific DNA sequences based on the amino acids that compose the portion of the protein that interfaces with the DNA. Most DNA-binding proteins interact with DNA as dimers. Sometime, each member of a dimer is specific for a particular DNA sequence, leading to heterodimerization. Heterodimerization of a leucine zipper: HELIX-LOOP-HELIX Two-stranded Leucine zipper dimer b-sheets dimer  GENETIC SWITCHES: Now that we are aware that gene regulatory proteins can interact with DNA through the structural motifs briefly discussed before, let’s consider some of the mechanisms of regulation, or … HOW DO GENETIC SWITCHES work ?  The tryptophan repressor: When bacteria have plenty of tryptophan available from the environment, they do not need to synthesize it; thus, the enzymes that synthesize tryptophan are not expressed. Transcription of the enzymes required for tryptophan biosynthesis is controlled by one single promoter, the genes (open reading frames) under the control of the promoter are collectively referred to as an OPERON (ORF A, B, C, D, and E):  The tryptophan repressor: When there is plenty of tryptophan in the cell, a repressor (DNA-binding protein) binds free tryptophan and becomes attached to the operator sequence in the middle of the promoter, preventing transcription of the tryptophan operon by RNA polymerase:  The tryptophan repressor: When tryptophan becomes scarce in the cell, the amino acid comes off the repressor, which changes conformation and falls off the operator sequence; now, RNA polymerase can bind the promoter and transcription of the tryptophan operon takes place:  The tryptophan repressor: The free amino acid tryptophan binds to the grooves of the REPRESSOR protein. The repressor becomes activated ONLY when tryptophan is attached and recognizes the operator sequence within the promoter and binds to it. The repressor binding to the OPERATOR prevents RNA polymerase to dock and initiate transcription of the tryptophan operon: This form of regulation of gene expression is called NEGATIVE CONTROL or REGULATION NEGATIVE REGULATION is mediated by REPRESSORS: REPRESSORS can modulate gene expression in two ways: 1. The repressor binds the operator sequence only in the absence of ligand and falls off when the ligand binds to it. OR 2. The repressor binds the operator sequence only in the presence of ligand and falls off when the ligand is removed (tryptophan repressor). Prokaryotic gene regulation POSITIVE REGULATION is mediated by ACTIVATORS: ACTIVATORS can modulate gene expression in two ways: 1. The activator binds the operator sequence only in the absence of ligand to stimulate RNA pol and falls off when the ligand binds to it. OR 2. The activator binds the operator sequence only in the presence of ligand to stimulate RNA pol and falls off when the ligand is removed. Prokaryotic gene regulation  In some cases, the exact location of the operator sequence determines whether a gene-regulatory protein acts as a repressor or an activator: In this example, the lambda protein acts as an activator, as its operator sequence is located behind the promoter. ® Transcription takes place. In this example, the same lambda protein acts as a repressor on another promoter, because the operator sequence overlaps with the promoter. ® Transcription does not take place. Prokaryotic gene regulation  Eukaryotic genetic switches: Eukaryotic cells assemble a large and complex number of transcription factors to mediate RNA polymerase transcription leaving little additional space for simple repressor and activator proteins as those used by bacteria. To overcome this “crowding problem”, gene regulatory proteins of eukaryotes can act when bound thousands of nucleotides away from the site of transcriptional start ! This means that a single promoter can be controlled by a virtually unlimited number of regulatory sequences scattered along the DNA. Because eukaryotes require this large set of proteins to assemble at the promoter, and these have to be assembled in a sequential manner, this provides within itself an opportunity to regulate transcription by affecting the steps involved in assembly of the transcription factors. Furthermore, the compaction into the tight chromatin structure provides additional opportunities for regulation not available to bacteria.  How does it work ? The basis for this mechanism is the ability of DNA to bend upon itself. The bending capability of The activator (in this case NtrC) binds a DNA determines the likelihood sequence of DNA called enhancer, several base two proteins bound to DNA will pairs upstream of the promoter sequence. meet: The DNA bends back and the activator interacts (touches) RNA polymerase thereby stimulating transcription: NtrC RNA pol DNA [This is a rare example of bacteria using enhancer sequences, this is a mechanism employed mostly by eukaryotes.]  Regulatory sequences of eukaryotic genes can be distant from the promoter where RNA polymerase starts transcription. Gene regulatory proteins recognize and bind to these regulatory sequences. DNA looping allows gene-regulatory proteins to interact with the transcriptional machinery (TFs and RNA Pol): While the transcriptional machinery is the same, the gene-regulatory proteins change from one gene to the other. Gene-regulatory proteins recognize specific DNA-binding sites: The Gal-4 protein recognizes the Gal-4 DNA-binding site and induces transcription by RNA polymerase. When a chimeric protein (a hybrid) is made by fusing the Gal-4 activation domain to the DNA-binding domain of the LexA protein, the chimera cannot bind the Gal-4 DNA binding site. However, the chimera recognizes and binds to the DNA-binding site for LexA inducing transcription by RNA polymerase. The DNA-binding domain ALONE of gene-regulatory proteins recognizes a specific DNA binding site.  BINDING OF A GENE-REGULATORY PROTEIN RECRUITS RNA POL AND TRANSCRIPTION FACTORS (collectively known as HOLOENZYME) TO THE PROMOTER: An activator finds and binds to the DNA-binding site upstream of the promoter This event leads to recruitment of transcription factors and the RNA POL HOLOENZYME Interaction of the activator with the HOLOENZYME leads to transcriptional activation.  Chromatin must be remodeled for the transcriptional machinery to access the DNA: Chromatin remodeling can be accomplished in two ways: 1. Nucleosome remodeling using CHROMATIN REMODELING COMPLEX. 2. Histone acetylation using HISTONE ACETYLASE (HAT). Both mechanism lead to localized alterations of the chromatin structure that allows for accessibility of DNA sequences by gene-regulatory proteins. Transcription factors are not able to assemble on a promoter if the DNA is packaged into nucleosomes. We have seen how eukaryotic genes can be activated, here are 5 ways a eukaryotic gene can be repressed: (A) A repressor binds to the DNA binding site preventing an activator to bind to its nearby site. (B) A repressor binds to the activation domain of the nearby activator. (C) Interaction of the repressor with the TFs preventing binding of the HOLOENZYME to the promoter. (D) A repressor recruits a chromatin remodeling complex to tighten the chromatin thereby “hiding” the promoter sequence. (E) A repressor attracts a histone deacetylase to the promoter region preventing TFIID binding to the promoter and keep the chromatin tightly wound. Gene-regulatory proteins must become activated to bind DNA: (A) Some proteins are synthesized (active) only when they are needed and rapidly degraded inside the cell. (B) Some proteins must bind a ligand to become activated (tryptophan repressor). (C) Some proteins must be phosphorylated to become active. (D) Some proteins must associate with a subunit to become activated. (E) Some proteins must remove (F) Some proteins become activated upon entry into an inhibitor to become the nucleus when they shed an inhibitor. activated. (G) Some proteins become activated when cleaved off the membrane Each one of these activation mechanisms is usually controlled by extracellular signals.  Thus far we have reviewed mechanisms prokaryotes and eukaryotes employ to regulate transcriptional initiation by mean of genetic switches. Next, we will review two mechanisms cells use to regulate expression of genes after transcription is complete (post- transcriptional regulation). Eukaryotes can use alternative splicing to produce several different proteins from one single gene: After transcription is complete, eukaryotes splice their RNA to remove introns. Alternative splicing allows for one single RNA molecule to be processed differently yielding separate mRNAs and therefore proteins depending on the introns removed. Alternative splicing may explain why some eukaryotic genomes have less genes than originally predicted. In each examples, a single RNA is spliced in two alternative ways to produce two different mRNAs and therefore proteins. The dark blue boxes show exon sequences that are retained by both mRNAs. The light blue boxes show exon or intron sequences that are included only in one mRNA. The boxes joined by red lines indicate where intron sequences (yellow) are removed. The DSCAM gene of fruit flies can be spliced into 38’016 variant mRNAs The DSCAM gene encodes a family of receptor proteins. The final mRNA is made up of 24 exons, but these can be made up of any combination of A, B, C, and D exons. The red line line shows the splicing pattern used to produce the mRNA shown. Although the potential exists for 38’016 different mRNAs (and therefore proteins) to be encoded by alternative splicing of the DSCAM RNA, only a few variants have actually been observed. Mammalian immunolglobulin genes also use sophisticated alternative splicing mechanisms giving rise to a large number of different antibodies each having unique antigen recognition properties. Gene expression can also be controlled TRANSLATIONALLY: !"#$%&#'()$*"+,"+%%)"*,")'+($%*-#$*"+-).$(/+*#$0*1($0* ')*%+-)$0#"2*%'"3-'3"+%*4)"5+0*#'*'6+*78*+$0*)4*'6+* 9:;*'6+"+12*,"+