MOLECULAR BIOLOGY AND DIAGNOSTICS GENE REGULATION OUTLINE • Molecular Definition of Gene Regulation o Introduction o Molecular Definition of Gene Regulation o Gene Regulation in Eukaryotes o Gene Regulation in Prokaryotes o Differences in the Regulation of Gene Expression of Prokaryotic & Eukaryotic Organisms o • Stages of Gene Regulation o Introduction o Transcription o Translation • Genomic Imprinting o Introduction • Transcriptional Regulation o Introduction o Transcriptional Regulation o Post-transcriptional Regulation • Examples of Eukaryotic Gene Regulation o Eukaryotic Gene Regulation o Gene Regulation in Development and Evolution L1: MOLECULAR DEFINITION OF GENE REGULATION • Gene expression • Protein synthesis INTRODUCTION CENTRAL DOGMA • Process in which the genetic information flows from DNA to RNA, to make a functional product protein • Transcription o Process by which the information is transferred from one strand of the DNA to RNA by the enzyme RNA polymerase o • Translation o Process by which the RNA codes for specific proteins o An active process which requires which requires energy. This energy is provided by the charged tRNA molecules o Ribosomes initiate the translation process. MOLECULAR DEFINITION OF GENE REGULATION • All cells control or regulate the synthesis of proteins from information encoded in their DNA. The process of turning on a gene to produce RNA and protein is called gene expression. The regulation of gene expression conserves energy and space. • There must be a mechanism to control when a gene is expressed to make RNA and protein, how much of the protein is made, and when it is time to stop making that protein because it is no longer needed. • It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on genes only when they are required. • The control of gene expression is extremely complex. Malfunctions in this process are detrimental to the cell and can lead to the development of many diseases, including cancer. • These different patterns of gene expression cause your various cell types to have different sets of proteins, making each cell type uniquely specialized to do its job. • Gene Turned On : How do these cues help a cell decide what genes to express? Cells don’t make decisions in the sense that you or I would. They have molecular pathways that convert information —such as the binding of a chemical signal to its receptor —into a change in gene expression o Gene regulation is the process of controlling which genes in a cells’ DNA are expressed (used to make a functional product such as a protein) GROWTH FACTOR PROMPTING CELL DIVISION 1. The cell detects the growth factor through physical binding of the growth factor to a receptor protein on the cell surface. 2. Binding of the growth factor causes the receptor to change shape, triggering a series of chemical events in the cell that activate proteins called transcription factors. 3. The transcription factors bind to certain sequences of DNA in the nucleus and cause transcription of cell division-related genes. 4. The products of these genes are various types of proteins that make the cell divide (drive cell growth and/or push the cell forward in the cell cycle). 5. Gene regulation is the process of controlling which genes in a cell’s DNA are expressed (used to make a functional product such as a protein) GENE REGULATION IN EUKARYOTES • Eukaryotic cells, in contrast, have intracellular organelles that add to their complexity. In eukaryotic cells, the DNA is contained inside the cell’s nucleus and there it is transcribed into RNA.
• The newly synthesized RNA is then transported out of the nucleus into the cytoplasm, where ribosomes translate the RNA into protein. • The processes of transcription and translation are physically separated by nuclear membrane; transcription occurs only within the nucleus, and translation occurs only outside the nucleus in the cytoplasm. • Eukaryotes: Nucleus. GENE REGULATION IN PROKARYOTES • Prokaryotic organisms are single-celled organisms that lack a cell nucleus, and their DNA therefore floats freely in the cell cytoplasm. • To synthesize a protein: the processes of transcription and translation occur almost simultaneously. When the resulting protein is no longer needed, transcription stops. As a result, the primary method to control what type of protein and how much of each protein is expressed in a prokaryotic cell is the regulation of DNA transcription. • All of the subsequent steps occur automatically. When more protein is required, more transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is mostly at the transcriptional level. • Prokaryotes: Cytoplasm DIFFERENCES IN THE REGULATION OF GENE EXPRESSION OF PROKARYOTIC & EUKARYOTIC ORGANISMS Prokaryotic Eukaryotic Lack nucleus Contain nucleus DNA is found in the cytoplasm DNA is confined to the nuclear compartment RNA transcription and protein formation occur almost simultaneously RNA transcription occurs prior to protein formation, and it takes place in the nucleus. Translation of RNA to protein occurs in the cytoplasm. Gene expression is regulated primarily at the transcriptional level. Gene expression is regulated at many levels (epigenetic, transcriptional, nuclear shifting, post-transcriptional, translational, and post-translational) L2: STAGES OF GENE REGULATION INTRODUCTION TWO MAIN STAGES • Transcription : the production of messenger RNA (mRNA) by the enzyme RNA polymerase, and the processing of the resulting mRNA molecule. • Translation : the use of mRNA to direct protein synthesis, and the subsequent post-translational processing of the protein molecule. • Some genes are responsible for the production of other forms of RNA that play a role in translation, including transfer RNA (tRNA) and ribosomal RNA (rRNA). • A structural gene involves a number of different components: • Exons - code for amino acids and collectively determine the amino acid sequence of the protein product. It is these portions of the gene that are represented in final mature mRNA molecule. • Introns - Introns are portions of the gene that do not code for amino acids, and are removed (spliced) from the mRNA molecule before translation. • Regulatory gene - regulates the genes • Regulatory sequences - found top or above the gene GENE CONTROL REGIONS • Start site - start site for transcription. • Promoter - A region a few hundred nucleotides 'upstream' of the gene (toward the 5' end). It is not transcribed into mRNA, but plays a role in controlling the transcription of the gene. Transcription factors bind to specific nucleotide sequences in the promoter region and assist in the binding of RNA polymerases. • Enhancers - Some transcription factors (called activators) bind to regions called 'enhancers' that increase the rate of transcription. These sites may be thousands of nucleotides from the coding sequences or within an intron. Some enhancers are conditional and only work in the presence of other factors as well as transcription factors. • Silencers - Some transcription factors (called repressors) bind to regions called 'silencers' that depress the rate of transcription.
TRANSCRIPTION • the process of RNA synthesis, controlled by the interaction of promoters and enhancers. Several different types of RNA are produced, including messenger RNA (mRNA), which specifies the sequence of amino acids in the protein product, plus transfer RNA (tRNA) and ribosomal RNA (rRNA), which play a role in the translation process. • Transcription involves four steps: 1. Initiation . The DNA molecule unwinds and separates to form a small open complex. RNA polymerase binds to the promoter of the template strand. 2. Elongation . RNA polymerase moves along the template strand, synthesizing an mRNA molecule. In prokaryotes RNA polymerase is a holoenzyme consisting of a number of subunits, including a sigma factor(transcription factor) that recognizes the promoter. In eukaryotes there are three RNA polymerases: I, II and III. The process includes a proofreading mechanism. 3. Termination . In prokaryotes there are two ways in which transcription is terminated. In Rho-dependenttermination, a protein factor called "Rho" is responsible for disrupting the complex involving the template strand, RNA polymerase and RNA molecule. In Rho-independent termination, a loop forms at the end of the RNA molecule, causing it to detach itself. Termination in eukaryotes is more complicated, involving the addition of additional adenine nucleotides at the 3' of the RNA transcript (a process referred to as polyadenylation). 4. Processing . After transcription the RNA molecule is processed in a number of ways: introns are removed and the exons are spliced together to form a mature mRNA molecule consisting of a single protein-coding sequence. RNA synthesis involves the normal base pairing rules, but the base thymine is replaced with the base uracil. • TRANSLATION • In translation, the mature mRNA molecule is used as a template to assemble a series of amino acids to produce a polypeptide with a specific amino acid sequence. The complex in the cytoplasm at which this occurs is called a ribosome. Ribosomes are a mixture of ribosomal proteins and ribosomal RNA (rRNA), and consist of a large subunit and a small subunit. • Translation involves four steps: 1. Initiation . The small subunit of the ribosome binds at the 5' end of the mRNA molecule and moves in a 3' direction until it meets a start codon (AUG). It then forms a complex with the large unit of the ribosome complex and an initiation tRNA molecule. 2. Elongation . Subsequent codons on the mRNA molecule determine which tRNA molecule linked to an amino acid binds to the mRNA. An enzyme peptidyl transferase links the amino acids together using peptide bonds. The process continues, producing a chain of amino acids as the ribosome moves along the mRNA molecule. 3. Termination . Translation in terminated when the ribosomal complex reached one or more stop codons (UAA, UAG, UGA). The ribosomal complex in eukaryotes is larger and more complicated than in prokaryotes. In addition, the processes of transcription and translation are divided in eukaryotes between the nucleus (transcription) and the cytoplasm (translation), which provides more opportunities for the regulation of gene expression. 4. Post-translation processing of the protein. L3: GENOMIC IMPRINTING INTRODUCTION • The expression of a small number of human genes is influenced by whether the gene has been inherited from the mother or father. This process - called genomic (or parental) imprinting - usually means that the organism expresses one of its alleles but not both. • Genomic imprinting is a reversible form of gene inactivation and is not considered a mutation. For instance, Jane inherits two copies of a paternally imprinted gene. The copy she inherited from her father will be imprinted, or inactivated, and the copy she inherited from her mother will be active (not imprinted). However, all the imprinted genes that Jane passes onto her offspring must be maternally imprinted. • Conversion of imprints occurs in each individual’s egg and sperm cells during their embryonic development, so that the genes they pass on reflect their gender, and not their parents.
• Each chromosome in an egg cell has a maternal imprint. Likewise, each chromosome in a sperm cell has a paternal imprint. The photo depicts one of the 23 chromosomes in each germline cell, and illustrates the process by which the maternal or paternal imprinting is converted to the proper imprinting for the next generation. • Following fertilization and conception, the resulting cell retains the imprints signifying from which parent each chromosome was inherited. • During embryonic development, the chromosomes in the cells of the somatic tissues of the body also retain the parental imprints. • However, in the gametes, or egg and sperm cells, the chromosomes must be converted to the gender of the new individual. Therefore, the chromosomes in a sperm cell will now all be paternally imprinted and those in an egg cell will be maternally imprinted. In other words, in the female the paternally imprinted chromosome is now maternally imprinted, and the maternally imprinted one in the male is now paternally imprinted. • L4: TRANSCRIPTIONAL REGULATION INTRODUCTION • Transcription is the process where a gene's DNA sequence is copied (transcribed) into an RNA molecule. Transcription is a key step in using information from a gene to make a protein. • The enzyme RNA polymerase, which makes a new RNA molecule from a DNA template, must attach to the DNA of the gene. It attaches at a spot called the promoter. • In humans and other eukaryotes, there is an extra step. RNA polymerase can attach to the promoter only with the help of proteins called basal (general) transcription factors. They are part of the cell's core transcription toolkit, needed for the transcription of any gene. • How do transcription factors work: A typical transcription factor binds to DNA at a certain target sequence. Once it's bound, the transcription factor makes it either harder or easier for RNA polymerase to bind to promoter of gene. TRANSCRIPTIONAL REGULATION • Activators • Repressor - may get in the way of the basal transcription factors or RNA polymerase, making it so they can't bind to the promoter or begin transcription. • Binding sites o The flexibility of DNA is what allows transcription factors at distant binding sites to do their job. The DNA loops like cooked spaghetti to bring far-off binding sites and transcription factors close to general transcription factors or "mediator" proteins. o The flexibility of DNA is what allows transcription factors at distant binding sites to do their job.
POST-TRANSCIPTIONAL REGULATION • Even after a gene has been transcribed, gene expression can still be regulated at various stages. • Some transcripts can undergo alternative splicing, making different mRNAs and proteins from the same RNA transcript. • Some mRNAs are targeted by microRNAs, small regulator RNAs that can cause an mRNA to be chopped up or block translation. • A protein's activity may be regulated after translation, for example, through removal of amino acids or addition of chemical groups. • The miRNA directs the protein complex to "matching" mRNA molecules (ones that form base pairs with the miRNA). When the RNA-protein complex binds. • Their direct role is to reduce the expression of their target genes, but they may play this role to produce many different outcomes L5: EXAMPLES OF EUKARYOTIC GENE REGULATION EUKARYOTIC GENE REGULATION GENE REGULATION IN DEVELOPMENT AND EVOLUTION • Prokaryotic cells can only regulate gene expression by controlling the amount of transcription. As eukaryotic cells evolved, the complexity of the control of gene expression increased. For example, with the evolution of eukaryotic cells came compartmentalization of important cellular components and cellular processes. A nuclear region that contains the DNA was formed. Transcription and translation were physically separated into two different cellular compartments. It therefore became possible to control gene expression by regulating transcription in the nucleus, and also by controlling the RNA levels and protein translation present outside the nucleus. • Some cellular processes arose from the need of the organism to defend itself. Cellular processes such as gene silencing developed to protect the cell from viral or parasitic infections. If the cell could quickly shut off gene expression for a short period of time, it would be able to survive an infection when other organisms could not. Therefore, the organism evolved a new process that helped it survive, and it was able to pass this new development to offspring.