Regulation of Gene Expression基因表达调控 Deqiao Sheng PhD Dept. of Biochemistry and Molecular Biology
Reference Books • Leningers’ Principles of Biochemistry • Harpers’ Biochemistry 26th edition • Styers’ Biochemistry • Hortons’Principles of Biochemistry4th edition
Basic conceptions Diagram of the central dogma, DNA to RNA to protein, illustrating the genetic code. Gene expression
A gene(基因) is the basic unit of heredity in a living organism. • All living things depend on genes. Genes hold the information to build and maintain an organism's cells and pass genetic traits to offspring. • Function units
Genome——is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. • E.coli contains about 4,400 genes present on a single chromosome • Human genome is more complex, with 23 pairs of chromosomes containing 6 billion(6×109) base pairs of DNA. 30,000~40,000 genes
Concept of gene expression • Gene expression is the combined process of the transcription of a gene into mRNA, the processing of that mRNA, and its translation into protein • Gene expressionis the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA.
The genetic information present in each somatic cell of a metazoan organism (multicellular animals ) is practically identical. How to meet the different needs? Different function need different proteins. • Regulated expression of genes is required for development, differentiation, and adaption.
In genetics gene expression is the most fundamental level at which genotype gives rise to the phenotype. • The genetic code is "interpreted" by gene expression, and the properties of the expression products give rise to the organism's phenotype. • Genotype→Phenotype
Genotype Information Flow • A gene is turned on and transcribed into RNA • Information flows from genes to proteins, genotype to phenotype Phenotype
The cellular concentration of a protein is determined by a delicate balance of at least seven processes, each having several potential points of regulation.
Points of Regulation • Transcription • Post-transcriptional modification • mRNA degradation rate • Translation • Post-translational modification • Protein targeting and transport • Protein degradation
Regulation of Gene Expression General • The regulation of the expression of genes is absolutely essential for the growth, development, differentiation and the very existence of an organism. • The are two types of gene regulation-positive and negative.
Positive regulation: the gene regulation is said to be positive when its expression is increased by a regulatory element (positive regulator) • Negative regulation: A decrease in the gene expression due to the presence of a regulatory element (negative regulator) is referred to as negative regulation.
The aim of the control To select the right gene To express at the right time To express at the right place What When Where The right gene expresses at the right time & the right place.
The significance of gene expression regulation • The differential transcription of different genes largely determines the actions and properties of cells. • Regulation at any one of the various steps in this process could lead to differential gene expression in different cell types or developmental stages or in response to external conditions (such as: Environments). • Temporal specificity (stage specificity) • Spatial specificity (cell or tissue specificity)
FROM EGG TO ORGANISM: HOW AND WHY GENES ARE REGULATED • Four of the many different types of human cells • They all share the same genome Genotype (DNA) • What makes them different? Phenotype (Protein) (a) Three muscle cells (partial) (b) A nerve cell (partial) One of underlying principles of molecular cell biology is that the actions and properties of each cell type are determined by the proteins it contains. (c) Sperm cells (d) Blood cells
One of the characters of gene expression : it is precisely controlled to be activated in the right cells and right time during development of the many different cell types that collectively form a multicellular organism. e.g. Human Hemoglobin(血红蛋白) • Human hemoglobin is consisted of two alpha-like and beta-like globin chains, which are coded by alpha-like and beta-like globin genes respectively.
Hemoglobin clusters • Human hemoglobin: (at developmental stages) • z2e2 • HbFa2g2(end of trimester) • HbAa2b2(start from the third trimester , do not completely replace g chains until some weeks postpartum)
Regulation of GeneExpression • Principles of gene regulation • Regulation of gene expression in prokaryotes • Regulation of gene expression in eukaryotes
Principles of Gene Regulation • Constitutive gene expression • A gene is expressed at the same level at all times. housekeeping gene • Regulated gene expression • Inducible :Gene products that increase in concentration under particular molecular circumstances. • Repressible: gene products that decrease in concentration in response to a molecular signal.
Constitutive genes • Constitutive genes: refer to genes whose expression are not regulated. The products of these genes are produced at a constant rate. Such genes are called constitutive genes and their expression is said to be constitutive. e.g. b-actin-Actins are highly conserved proteins that are involved in cell motility, structure and integrity. GAPDH (glyceraldehyde-3-phosphate dehydrogenase )-is an enzyme that catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules.
Products of the constitutive genes are required at all times, such as those for the enzymes of central metabolic pathways. Those genes are expressed at a more or less constant level in virtually every cell of a species or organism. They are often referred to as housekeeping genes also.
Inducible genes • Inducible genes refer to the genes whose expression increases in response to an inducer, a specific regulatory signal. The process is called induction. e.g. The expression of many of the genes encoding DNA repair enzymes, for example, is induced by high levels of DNA damage.
The Structure of Gene • Structural gene • codes for a protein (or RNA) product • Regulatory gene • codes for a protein (or an RNA) involved in regulating the expression of other genes
Structural and Regulatory gene • A structural gene : Structural genes represent an enormous variety of protein structures and functions, including structural proteins, enzymes and regulatory proteins. • A regulatory gene : The interaction can regulate a target gene in a manner either positive (the interaction turns the gene on) or negative (the interaction turns the gene off).
RNA Polymerase Binds to DNA at Promoters • RNA polymerases bind to DNA and initiate transcription at promoters , sites generally found near points at which RNA synthesis begins on the DNA template. • The nucleotide sequences of promoters vary considerably, affecting the binding affinity of RNA polymerases and thus the frequency of transcription initiation.
Sequences of promoters • Consensus sequence for many E. coli promoters. (procaryotic) • -10 regionTATAAT • -35 regionTTGACA • Most base substitutions in the -10 and -35 regions have a negative effect on promoter function. Some promoters also include the UP (upstream promoter) element
Transcription activity ? • promoter sequence • Mutations that result in a shift away from the consensus sequence usually decrease promoter function; conversely, mutations toward consensus usually enhance promoter function. • regulatory proteins • It can modulate non-housekeeping genes expression
Common sequences in promoters recognized by eukaryotic RNA polymerase II. • -30 region TATA box • Initiator sequence (Inr) N , represents any nucleotide Y, a pyrimidine nucleotide
RNA Polymerase II Requires Many Other ProteinFactors for Its Activity • specificity factors • Alter the specificity of RNA polymerase for a given promoter or set of promoters • repressors • impede access of RNA polymerase to the promoter • activators • Enhance the RNA polymerase–promoter interaction.
RNA polymerase II holoenzyme complex bound to a promoter Transcription machinery
There are a lot of proteins participate in the regulation of gene expression. • Transcription Factor (TF) • Activators • Repressors • Regulatory proteins
Specificity factors • Prokaryotic specificity factors • The subunit of the E. coli RNA polymerase holoenzyme is a specificity factor that mediates promoter recognition and binding. • Eukaryotic specificity factors • the TATA-binding protein (TBP)
Repressors • Protein • Bind to specific sites on the DNA • In prokaryotic cells, such binding sites, called operators, are generally near a promoter. • Blocks transcription/negative regulation
Activators • Activators provide a molecular counterpoint to repressors; they bind to DNA and enhance the activity of RNA polymerase at a promoter • positive regulation • binding sites are often adjacent to promoters that are bound weakly or not at all by RNA polymerase alone
Enhancers • positive regulation • Some eukaryotic activators bind to DNA sites, called enhancers, that are quite distant from the promoter, affecting the rate of transcription at a promoter that may be located thousands of base pairs away. • Some activators are normally bound to DNA, enhancing transcription until dissociation of the activator is triggered by the binding of a signal molecule .
Regulatory proteins • Three domain (at least two) • DNA binding domain • Bind to DNA • protein-protein interaction domain • Interact with RNA polymerase, other regulatory proteins, or other subunits of the same regulatory protein. • dimerization domain Domain—An independently folded part of a protein.
Within regulatory proteins, the amino acid side chains most often hydrogen-bonding to bases in the DNA are those of Asn, Gln, Glu, Lys, and Argresidues. • To interact with bases in the major groove of DNA, a protein requires a relatively small structure that can stably protrude from the protein surface.
DNA-binding sites • The DNA-binding sites for regulatory proteins are often inverted repeats of a short DNA sequence (a palindrome) at which multiple (usually two) subunits of a regulatory protein bind cooperatively. • The Lac repressor is unusual in that it functions as a tetramer, with two dimers tethered together at the end distant from the DNA-binding sites.
Relationship between the lac operator sequence and the lac promoter. AATTGT…ACAATT TTAACA…TGTTAA palindrome
DNA binding domain : DNA-binding sites :a short DNA sequence (a palindrome) • helix-turn-helix • zinc finger • homeodomain—found in some eukaryotic proteins.
helix-turn-helix • This DNA-binding motif is crucial to the interaction of many prokaryotic regulatory proteins with DNA, and similar motifs occur in some eukaryotic regulatory proteins. • The helix-turn-helix motif comprises about 20 amino acids in two short a -helical segments, each seven to nine amino acid residues long, separated by a b turn
Helix-turn-helix DNA-binding domain of the Lac repressor. The helix-turn-helix motif is shown in red and orange; the DNA recognition helix is red.
Zinc Finger • In a zinc finger, about 30 amino acid residues form an elongated loop held together at the base by a single Zn2+ ion, which is coordinated to four of the residues (four Cys, or two Cys and two His). • The zinc does not itself interact with DNA; rather, the coordination of zinc with the amino acid residues stabilizes this small structural motif. Several hydrophobic side chains in the core of the structure also lend stability.
Zinc fingers. Three zinc fingers (gray) of the regulatory protein Zif268, complexed with DNA (blue and white) . Each Zn2+ (maroon) coordinates with two His and two Cys residues (not shown).
Homeodomain • Another type of DNA-binding domain has been identified in a number of proteins that function as transcriptional regulators, especially during eukaryotic development. • This domain of 60 amino acids—called the homeodomain, because it was discovered in homeotic genes (genes that regulate the development of body patterns)—is highly conserved and has now been identified in proteins from a wide variety of organisms, including humans . The DNA-binding segment of the domain is related to the helix-turn-helix motif. The DNA sequence that encodes this domain is known as the homeobox.
Homeodomain. Shown here is a homeodomain bound to DNA; one of the helices (red), stacked on two others, can be seen protruding into the major groove . This is only a small part of the much larger protein Ultrabithorax (Ubx), active in the regulation of development in fruit flies.
Motif— • An independent folding unit, or particular structure, that recurs in many molecules. (DNA or protein) • Domain— • An independently folded part of a protein.
protein-protein interaction domain: Mediate interaction with RNA polymerase, other regulatory proteins, or other subunits of the same regulatory protein. • leucine zipper • basic helix-loop-helix.