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Page 1 of 16 Chapter 31 Notes Biochemistry 461 Fall 2010 CHAPTER 31: CONTROL OF GENE EXPRESSION LECTURE TOPICS ! Differential Gene Expression in Procaryotes and Eucaryotes ! Regulation of Gene Expession ! Transcriptional Regulation by DNA-Binding Proteins (E.coli Lactose Operon) ! Transcriptional and Postranscriptional Regulation (trp Operon Attenuation; ferritin/transferrin receptor in eucaryotes) ! Helix-turn-helix motif of Procaryotic DNA Binding Proteins ! Eucaryotic Gene Regulation ! Complexity of Genomes ! Elaborate Mechanisms of Regulation ! Transcription Activation/Repression mediated by Protein-Protein Interactions OVERVIEW: PROCARYOTIC GENE EXPRESSION ! Once again - Bacteria (E. coli) are most well studied with regard to regulation of gene expression. Differences in rates of synthesis of some proteins varies over a 1000-fold range in response to changes in quantity and quality of metabolites, nutrients, environmental challenges, etc. ! Regulation of bacterial gene expression occurs most often at the level of transcription. Genes are often clustered in operons, all of whose genes are transcribed coordinately in a single mRNA molecule. These genes can be expressed at all times in the cell (constitutive genes) or they can be under the control of repressor and/or activator proteins (inducible genes). Page 2 of 16 ! Many regulatory proteins which bind to DNA have a helix-turn-helix-motif in common. ! Examples of operons whose transcriptional regulation is well known are the lactose and tryptophan operons of E. coli. ! Gene expression is also regulated at the translational level. [Exs.: trp operon in E. coli; ferritin and transferrin receptor in eucaryotes. OVERVIEW: EUCARYOTIC CHROMOSOMES AND GENE EXPRESSION ! Eucaryotic chromosomes are larger, have higher degrees of structural order, and a more complex composition than procaryotic chromosomes. The human genome (4x109 bp = 1 meter) is 1,000 times the size of that of E. coli. DNA (4x106 bp = 1.4 mm). Replication and gene expression is more complex than the procaryotic model. ! Eu caryotic chromosomal DNA is wound around histones in complexes called nucleosomes. The entire chromosome also contains many other proteins in a complex matrix called chromatin. ! A small fraction (1- 2%) of eucaryotic DNA is genes which code for proteins. There is an abundance of repetitive, non-coding sequences which comprise a significant fraction of eucarotic genomes. ! Eucaryotic gene expression is regulated primarily at the level of transcription. Transcriptionally active regions of chromosomes are extrasensitive to DNase digestion and have reduced levels of cytosines which have been methylated. Expression of genes in these chromosomal regions is regulated by transcriptional factors. Page 3 of 16 ! Translational regulation occurs in iron metabolism genes. ! Some developmentally-controlled genes in insects and mammals possess a sequence called the homeo box, which codes for a 60 amino acid long DNAbinding polypeptide domain (homeo domain) which may be involved in regulating expression of these genes. DIFFERENTIAL GENE EXPRESSION IN PROCARYOTES AND EUCARYOTES ! Some procaryotic genes are differentially expressed in response to different environmental variables (temperature, nutrients, etc) ! Many genes are expressed at different times and in different locations (tissues, organs) in a eucaryotic organism during it’s lifespan. (Table 31.1) Page 4 of 16 REGULATION OF PROCARYOTIC GENE EXPESSION TRANSCRIPTIONAL REGULATION BY DNA-BINDING PROTEINS: E.coli Lactose Operon ! To use lactose as a sole carbon source, E. coli synthesizes $-galactosidase (thousands/cell) only when cells are grown on lactose. The enzyme is inducible by conversion in the cell of lactose to allolactose (a product of a reaction catalyzed by the 10 or so $-galactosidase molecules present in the cell prior to induction). (p.868 and Fig.31.1) $ - galactosidase induction: (Lactose or IPTG in lab) To measure $-galactosidase activity in the lab: X-gal cleavage gives galactose and a blue colored product that cna be quantitated. (Fig.31.1) Page 5 of 16 ! The $-galactosidase gene is in an operon which contains three structural genes (z, y, a), two control sites called the promoter (p) and operator (o), and a regulatory gene (i). Since there is more than one structural gene (cistron), the lactose operon is called a polycistronic operon. (Fig.31.3) ! The regulatory gene produces a repressor protein that normally (no lactose in medium) binds to the operator, preventing RNA polymerase from transcribing genes z, y, and a. An inducer(allolactose or IPTG), forms a repressor-inducer complex that cannot bind to the operator, permitting transcription. (Fig.31.8) Page 6 of 16 ! The lac repressor binds to an inverted repeat (operator) that is nearly a palindromic sequence with dyad symmetry. [Fig. 36-9] ! lac repressor DNA interactions (Figs.31.5, 6) ! An activator (CAP) protein binds cyclic AMP (levels increase when glucose in medium is low) and this cAMP-CAP complex bind upstream of the promoter site and stimulates transcription (50X more than without CAP). This overcomes the lac's lack of a strong promoter consensus sequence. (Fig.31.10) [Fig. 36-13] ! CAP-RNA polymerase binding sites are adjacent. RNA polymerase-repressor binding sites overlap. Repressor sterically interferes with RNA polymerase binding, while CAP facilitates binding of RNA polymerase. A CAP dimer (with cAMP) binds to DNA in the major groove and bends the DNA by 94 degrees!!! (Fig.31.11) Page 7 of 16 HELIX-TURN-HELIX MOTIF OF GENE REGULATING PROCARYOTIC DNA BINDING PROTEINS ! Three dimensional models of cro, lambda repressor, and CAP show that these polypeptides share an "-helix-$-turn-"-helix motif which is involved in specific protein-DNA interactions. These proteins occur as dimers which interact with specific DNA sequences via binding of an "-helical polypeptide domain with the major groove of a symmetrically-oriented recognition site spanning one turn of a B-DNA helix. [Fig. 36-1,27,29] ! $-strand DNA interactions are basis for recognition in methionine repressor (Fig.31.13) (recall also eucaryotic TATAbox binding protein) Page 8 of 16 PROCARYOTIC TRANSCRIPTIONAL AND POSTRANSCRIPTIONAL REGULATION TRYPTOPHAN OPERON REGULATION: ATTENUATION ! Transcription and translation interact to regulate the tryptophan operon. ! The tryptophan operon has several structural genes for proteins involved in tryptophan synthesis. There is an operator site where tryptophan repressor, complexed with tryptophan (a corepressor), binds, inhibiting transcription. [Figs. 36-31,32] ! A leader (L) sequence is 5' to an attenuator sequence [Fig. 36-34]. The leader codes for a polypeptide [Fig. 36-35] whose synthesis occurs at high cellular tryptophan levels and whose synthesis is inhibited at low tryptophan levels. The leader polypeptide has two tryptophan codons. (Fig.31.34) ! When cellular tryptophan concentration is high, leader translation is enhanced and transcription is inhibited by the attenuator's secondary structure, which looks like that of a terminator (i.e., a GC-rich region with two-fold symmetry followed by U's in the mRNA which makes a stem-loop structure). (Fig.31.34) [Fig. 36-36] ! When tryptophan levels are low, leader translation is slow, the tryp mRNA does not assume a terminator-like appearance, and the tryptophan operon is transcribed (Fig.31.35) [Fig. 36-36]. Trp Operon Attenuation Mechanism High Trp (W) fast translation 3-4 stem/loop stops transcription Low Trp (W) slow translation 2-3 stem/loop allows transcription Page 9 of 16 TRANSLATIONAL REGULATION IN EUCARYOTES: Proteins synthesis from ferritin and transferrin receptor mRNAs (Figs.31.38,39) ! IRE (iron response element) binding protein blocks translation of ferritin mRNA. ! Transferrin receptor mRNA has several IREs at 3'-end. IRE-binding protein located on these IRE’s stabilizes mRNA and does not inhibit translation. Page 10 of 16 EUCARYOTIC GENE REGULATION COMPLEXITY OF GENOMES: EUCARYOTIC CHROMOSOMES ! SIZE: Large genomes (1 meter long in human genome) linear molecules. (Figs.31.14.15) [Table 37-1] ! COMPOSITION: Contain five types of basic proteins called histones which have lots (25%) of Arg and Lys residues and are 11 to 21 Kd in mass. The histones are frequently modified by acetylation, methylation, Phosphorylation, etc. These modifications may relate to DNA packaging or availability for replication or transcription [Table 37-2]. Histone amino acid sequences and structures are highly conserved (especially histones H3 and H4) in all eucaryotes, suggesting that the role of histones was established early during eucaryotic evolution. (H2, 3, 4 structures, Fig.31.17) STRUCTURE: Page 11 of 16 ! Nucleosomes [Fig. 37-5, 8] are repeating units of chromatin which consist of core particles (a histone octamer around which is wrapped 140 base pairs of DNA) connected by linker DNA (20 to 50 base pairs). The core particles contain two molecules each of histones H2A, H2B, H3 and H4 and one molecule of histone H1 binds to the outside of the core particle. The DNA wound around the core particle forms a 1-3/4 turn left-handed superhelix [Fig.31.16, Fig. 33-8]. Nucleosomes condense DNA into a smaller space (packing ratio 7:1). Further DNA packing must occur in cells, since metaphase chromosomes have a packing ratio of 1000:1. (Fig.31.18) Page 12 of 16 ! A protein scaffold of non-histone proteins provides a higher order structure and higher packing ratio than that of just nucleosomes. These proteins include topoisomerase II which suggests that changes in supercoiling are important in dynamic changes of in chromosomal DNA structure and function during the cell cycle. [Fig. 37-11, and Lehninger, Fig. .23-20] Page 13 of 16 REPETITIVE VS. SINGLE COPY DNA ! Renaturation analysis revealed that eucaryotic chromosomal DNA has a large fraction of several types sequences (satellites, Alu sequences) that are repeated up to a million times. Results also showed that there was only a small fraction of unique protein-coding sequences. [Table 37-6] ! Ribosomal RNA genes are tandemly repeated several hundred times and can be amplified to 2X106 copies during development of Xenopus oocytes to up to 75% of total cellular DNA. [Amplification provides enough genes to rapidly transcribe rRNA genes to get 1012 ribosomes/oocyte.] ! Histone genes are clustered, repeated, lack introns, and have mRNA which is not polyadenylated. ! Many major (and minor) cell proteins are coded by single copy genes. For major cellular proteins, transcription rates, mRNA half-life, and use of mRNA for multiple rounds of translation can give up to 109 protein molecules from just one gene copy (silk fibroin). ! the human genome is 1000X the size of the E. coli chromosome, but only 1-2% codes for proteins. Thus, the actual unique human genome size is only about 50X that of E. coli. Page 14 of 16 REGULATION OF EUCARYOTIC GENE EXPRESSION: Elaborate Mechanisms ! Regions of chromosomes which are transcriptionally active are less condensed (form puffs in Drosophila [Fig. 37-29] are undermethylated, and are hypersensitive to DNase I (mostly at the 5'-ends of genes). These characteristics are tissue-specific and developmentally regulated. ! Methylation of cytosines [Fig. 37-30] in chromosomal DNA is associated with low transcriptional activity, in general. Azacytosine prevents methylation, so transcrption is more active. ! Not all possible regulatory sites on chromosomes in a given cell/tissue type may be activated at the same time. For instance, yeast GAL4 protein activates genes for proteins required in galactose utilization as a carbon source. But, of 4000 possible specific binding sequences, only 10 actually have GAL4 bound and only 10 genes must be activated for galactose utilization. Page 15 of 16 ! Disruption of chromatin structure occurs when transcription activator proteins bind to enhancer sequences. Many enhancer sequence elements are known. The glucocorticoid enhancer is one example (Ch.28 Notes, p.13). The muscle creatine kinase gene is another in which 3 copies of one enhancer sequence occurs and two other enhancer sequences exist. At least 3 different regulatory proteins are required to bind to all three of these different enhancers. Often, binding of these proteins disturbs the chromatin structure to expose the gene DNA to allow transcription. (Figs.31.20,21) TRANSCRIPTION ACTIVATION/REPRESSION MEDIATED BY PROTEIN-PROTEIN INTERACTIONS ! TRANSCRIPTIONAL ACTIVATORS: We already know about transcription factors and enhancer sequences (see Chapter 28). Transcriptional activators bind to specific activator sequences (enhancers) and these proteins have in common at least two conserved functional domains - one which binds to DNA and one which activates transcription (Fig.31.32; Fig.37-31] In addition, other conserved domains may be present, depending on the specific activator. Page 16 of 16 ! The family of nuclear hormone binding receptors (active as dimers of identical subunits) has an additional ligand-(hormone) binding domain. The DNAbinding domains of nuclear hormone receptor proteins possess globular structural domains in which four cysteines are tetrahedrally coordinated with a divalent zinc ion. Two of these zinc clusters are present on each subunit and they stabilize the structure of of the dimer. Each subunit of the dimeric steroid receptor has an "-helix which recognizes and binds to the major groove of the DNA sequence of the steroid response element. (Fig.31.22) Fig. 37-35 Fig.31-22 Fig. 37-36