Transcription Regulation 2 PDF

TRANSCRIPTIO N 1  TRANSCRIPTION Transcription: process of copying base sequence from DNA to complementary RNA Enzyme RNA polymerase reads nucleotides from DNA template in a 3’ to 5’ direction It assembles the complementary transcript in a 5’ to 3’ directio...

TRANSCRIPTIO N 1  TRANSCRIPTION Transcription: process of copying base sequence from DNA to complementary RNA Enzyme RNA polymerase reads nucleotides from DNA template in a 3’ to 5’ direction It assembles the complementary transcript in a 5’ to 3’ direction. Since it is synthesizing an RNA strand, it is pairing Uracil (U) with Adenine (A), instead of Thymine (T). mRNA synthesis is initiated when RNA Polymerase binds at a promoter sequence on DNA template Transcription continues (elongation) until RNA Pol reaches a stop or terminator sequence at end of gene Termination frees mRNA by the formation of a hairpin 2  CONCURRENT After termination, the process of transcription is complete. By the time termination occurs, prokaryotic transcript would have already begun synthesis of numerous copies of encoded protein because processes occur at same time. Transcription, translation and mRNA degradation are possible because they all occur in the same 5’ to 3’ direction and because there is no membrane compartment separating the processes in the nucleoid. Multiple polymerases can transcribe a single bacterial gene Multiple ribosomes can translate mRNA transcripts into polypeptides As a result, a specific protein can rapidly reach high concentrations in bacterial cell 3  OVERVIEW Before pre-mRNA is translated into protein, it is modified by undergoing splicing Splicing: introns sequences are removed & exons, which encode proteins and will be expressed are reconnected to form final mRNA Some pre-mRNA molecules are cut and spliced in different ways in different tissues. 1 Gene can produce many different mRNA molecules 4 TRANSLATION 5  OVERVIEW After info in gene has gone from pre-mRNA to mRNA it is ready to be translated into a polypeptide Ribosomes consist of small and large subunits of protein and rRNA, which bind mRNA. Many ribosomes can work on same mRNA at a time. Translation begins at initiating AUG on mRNA, specifying 1st amino acid in polypeptide: methionine Each amino acid is carried to ribosome by attaching to specific molecule of tRNA tRNA has amino acid attachment site at 1 end & anticodon sequence that interacts with mRNA codon through complementary base pairing at other end. Amino-acid charging enzymes ensure correct amino acid attached to correct tRNA. Sequential amino acids are linked by peptide bonds. mRNA is translated, undergoing elongation until stop/nonsense codon is reached. A release factor dissociates components and frees new polypeptide. Folding of protein occurs during and after translation. Once polypeptide is synthesized, its role as a protein is established. 6  PROTEIN SYNTHESIS MACHINERY Synthesis of proteins consumes more energy than any other metabolic process After water, proteins make up most of the mass in a cell as they perform every function for it. Translation involves decoding mRNA into amino acids (50-1000) that are covalently bonded, peptide bonds to create proteins. Each amino acid has an amino (NH2) + carboxyl (COOH) group. This reaction is catalyzed by ribosomes and generates 1 H2O (Hydrolysis or Dehydration) 7  TRANSLATION Ribosome attaches to an mRNA molecule in cytoplasm As each codon pass through ribosome tRNA brings amino acid Ribosome attaches amino acids to growing chain Each tRNA carries just 1 kind of amino acid Each tRNA has 3 unpaired bases called anticodon Anticodon is complementary to a codon on mRNA Ribosome has 2nd binding site for tRNA of next codon Ribosomes form covalent bond called peptide bonds between amino acids to create a polypeptide. Chain continues to grow until reach stop codon releasing mRNA and polypeptide 8  TRANSLATION R 9  3 TYPES OF RNA INVOLVED IN TRANSLATION mRNA carries coded message that directs process from nucleus to ribosomes tRNA molecules deliver amino acids to ribosomes to carry out translation Ribosomes are made up of proteins and rRNA molecules which carry out reactions of joining amino acids together. Overall RNA carries the genetic code & translates it too Gene codes for traits that make up a characteristic Proteins can be enzymes which catalyze a chemical reaction Catalyze: start or speed up chemical reaction by lowering the Ea Overall proteins build or operate components of living cells. 10 REGULATION 11  INTRO 12 For cell to function properly, necessary proteins must be synthesized from information encoded in DNA at the proper time. Regulation of gene expression conserves energy and space because it requires DNA to be unwound from coiled structure. Gene expression: regulated process of turning on a gene to produce RNA, which will be converted into a protein. Control when its expressed, how much its made and when its time to stop Similar genetic organisms have differences because genes are turned on at different times during development or/and responses to different environmental conditions. Malfunctions in gene expression can lead to diseases.  PROKARYOTES 13 DNA binding proteins regulate genes by controlling transcription Every gene must be expressed as they are unicellular, but not at same time. Operon: group of genes regulated together  Lac OPERON (INDUCER) 14 Inducible operons: have proteins that bind to activate or repress transcription depending on local environment and needs of cell. Lac Operon encodes genes necessary to acquire and process lactose from envr. Before lac operon is activated/transcribed, glucose level must be very low and lactose must be present. If lactose absent, repressor binds to operator to prevent transcription Energetically efficient as wasteful to produce proteins to process lactose if glucose readily available. Promoter (P): site where RNA polymerase can bind to begin transcription Operator (O): DNA-binding protein lac repressor attaches to DNA  Lac OPERON (INDUCER) 15 Lac Repressor blocks transcription when lactose is not present. It binds the O region and RNA polymerase cannot reach genes This binding causes operon/genes to turn off When lactose is present, it binds the Lac Repressor causing a shape (conformational) change which makes it fall off DNA. RNA polymerase can now bind to P site and transcription can proceed. As a result, operon/genes are turned on. mRNA generated from this process is translated into proteins that allow lactose to be broken down as an energy source for cell  PRO VS EUKARYOTIC GENE EXPRESSION 16 Prokaryotes are single-cell organisms. They lack a nucleus so DNA floats in cytoplasm region called nucleoid. Transcription and translation occur simultaneously. Primary method to control what type of protein and how much of it is expressed is through the regulation of DNA transcription. Eukaryotes have nuclear envelope separating transcription and translation. Epigenetic level: when DNA uncoiled and loosened from nucleosomes to bind transcription factors Transcriptional level: when RNA is transcribed. Post-transcriptional level: when RNA is processed and exported to cytoplasm Post translational level: after protein has been made  TRANSCRIPTION FACTORS 17 TATA box: short sequence of DNA found 30 base pairs before start of gene (upstream) It binds a protein (Transcription factors) that helps position RNA polymerase to begin the process. Transcription Factors: DNA-binding proteins that play an important role in regulating gene expression. Open tightly packed (Heterochromatin) to allow RNA polymerase to bind Block access to certain genes like repressor proteins Multiple may be needed to bind Enhancer region upstream of TATA box factors to start transcription Regulation can also occur by: Exit of mRNA from nucleus Stability of mRNA Breakdown of gene’s protein products  DEVELOPMENT 18 Gene Regulation more complicated on Eukaryotes because most are multi-cellular organism. Determines type and amount of protein produced. Differentiation: Genes expressed based on type of cell/organ. Structure = function Most cells carry the full genetic material (genome DNA) in nucleus, but do not use all of it. Homeotic Genes: Regulates organs that develop in specific parts of body Homeobox genes: code for transcription factors that activate other genes important in development and differentiation Hox genes: group of homeobox genes located next to each other  EPIGENETICS 19 Homeotic Genes: Regulates organs that develop in specific parts of body Master control genes are switches that trigger patterns of development and differentiation, and they exist between species indicating common ancestry DNA is supercoiled by protein called histone, forming a cluster called nucleosomes which leads to structure called chromatin. Large # of methyl groups lead to condensed chromatin (Heterochromatin) which block expression as RNA polymerase cannot attach. Large # of acetyl groups lead to lose chromatin (Euchromatin) Epigenetics: process that alters genes without affecting nucleotide sequence. Ex. Methylation that changes how they are expressed.  PRO VS EUKARYOTIC GENE EXPRESSION 20 A  OTHER FACTORS 21 Environmental factors such as temperature, salinity and nutrient availability can also regulate gene expression. Children of malnourished mother may encounter higher rates of obesity when developed into adults During fetus stage the epigenetic changes altered the body so it was better suited for food scarcity. After war, when food is plentiful, organism’s health is negatively impacted because may not be able to change this mechanism. CANCER 22  OVERVIEW 23 Cancer is a disease of altered gene expression that can occur at every level of control, including at levels of DNA methylation, histone acetylation and activation of transcription factors. Changes in activity of tumor suppressor gene can result in cancer. Cancer is not a single disease. Mutations modify cell-cycle control and cells do not stop growing. For cells to move through each cell cycle, they must pass through checkpoints that ensure that each step is completed properly and no mutation that would alter function took place. Cyclin is a protein that serves as a checkpoint. Its phosphorylation allows cells to progress unimpeded through cycle contributing to the development of cancer.  ALTERED GENE EXPRESSION 24 A gene that is not normally expressed in a cell can be switched on as a result of gene mutation or changes in regulation (epigenetic, transcription, post transcription, translation or post-translation). Can detect changes at different levels when observing cancer: Epigenetics: changes in histone acetylation leading to gene silencing Activation of transcription factors by phosphorylation Increase RNA stability Increased translational control Protein modification  TUMOR SUPPRESOR GENES 25 Tumor suppressor genes: in normal cells, genes that function to prevent excess growth. P53 is a transcription factor. Mutated in over 50% of all cancers. It can bind to sites of genes to initiate transcription. Proto-oncogenes: positive cell-cycle regulators. Mutated form called oncogenes. Overexpression leads to uncontrolled cell growth. They can alter transcriptional activity, stability or protein translation of another gene. Myc is TF that is activated in Lymphoma, transforming B cells into cancerous cells that grow uncontrollably. Can result in tumors that interfere with normal functions.  CANCER & EPIGENETIC 26 In cancer cells, DNA in promoter region of silence genes is methylated on cytosine DNA residues in CpG islands. Histone proteins that surround that region lack acetyl modification present in normal cells that normally express that region. These changes are temporary and can be reversed. As a result, can design medications that can: Prevent histone deacetylase from removing acetyl group Prevent DNA methyl transferase from adding methyl groups to cytosines  CANCER & TRANSCRIPTION 27 Mutations that activate transcription factors with phosphorylation can increase binding of TF to binding site in a promoter. Activated gene could be linked with modified cell growth Mutation in DNA of promoter or enhancer region can increase binding ability of TF. Could lead to overexpression of gene resulting in cancer. In subset of breast cancer, epidermal growth factor receptor (EGFR) activates protein kinases that activate many TF through phosphorylation. Medications being developed to prevent activation of EGFR to treat these types of cancer.  CANCER & TRANSLATIONAL 28 Modifications in cancer cells can include increased translation of a protein, changes in phosphorylation, or alternative splice variants. Colon cancer involves Long (c-FLIPL) & short (c-FLIPS) proteins, which initiate controlled cell death mechanisms in normal cells. An increase in long form results in an increase cell growth instead of death. The expression of the wrong protein alters cell function and contributes to the development of cancer.  TARGET THERAPIES 29 Using what is known about regulation of gene expression, new medications can be developed to treat and prevent diseases such as cancer. If find a way to target a specific tumor, can result in less invasive, more effective and less detrimental treatments that do not harm healthy cells. As a result, therapy & medicines tailored to individuals has given rise to personalized medicine $$$ Targeted therapies: medications that exploit overexpression of a specific protein or the mutation of a gene to treat disease.