Gene Expression

GENE EXPRESSION GENES Genes are sequences of DNA that encode a specific characteristic (via the production of RNA or protein). They function as the basic units of inheritance. Genes occupy specific positions on chromosomes (loci). Genes can be categorised as being either structural or regulatory: • Structural genes: Code for proteins that contribute to either the form or function of the cell • Regulatory genes: Code for transcription factors that control gene expression (turns genes on or off) STRUCTURE OF A GENE A gene is composed of three key sections. The promoter is the site to which the enzyme RNA polymerase will bind – it is responsible for initiating transcription. The coding sequence is the region of DNA that is transcribed into RNA, while the terminator sequence functions to stop transcription by RNA polymerase. Basically, genes consist of start regions (promoter), copying regions (coding) and stop regions (terminator). Promoter Coding Sequence Terminator 5’ 3’ 3’ 5’ TRANSCRIPTION Transcription is the process by which a DNA sequence (gene) is copied into complementary RNA sequences by RNA polymerase. This enzyme binds to the promoter and then separates the double-stranded DNA of the coding sequence (by breaking the hydrogen bonds between base pairs). Free RNA nucleotides then align opposite their complementary base partner and RNA polymerase joins them together with covalent bonds (between the sugar-phosphate backbone). When the enzyme reaches the terminator sequence the synthesised RNA transcript is released and the double helix reforms. Transcription occurs in the nucleus. POST-TRANSCRIPTIONAL MODIFICATIONS In eukaryotic cells, there are three post-transcriptional modifications that must occur in order to convert the RNA transcript into messenger RNA (mRNA) that is then capable of being translated by the ribosome: • • • Capping: A methyl group is added to the 5’-end of a transcript (prevents degradation by exonucleases) Polyadenylation: A poly-A tail is added to the 3’-end of a transcript (improves transcript stability) Splicing: Non-coding sequences (called introns) must be removed from within the eukaryotic gene SPLICING Introns are intruding sequences within eukaryotic genes that do not code for protein, whereas exons are the expressing sequences within the gene that encode the relevant amino acid sequence. Prokaryotic cells do not typically possess introns and hence do not require splicing of the RNA transcript prior to translation. It is possible to remove specific exon sequences from a gene to create different forms of the same protein (e.g. removing an exon could determine whether a protein is membrane-bound or found in the cytosol). The differential removal of exon segments is called alternative splicing. Eukaryotic genes without introns can be artificially synthesised by converting mRNA into a DNA sequence via the enzyme reverse transcriptase (from retroviruses). The intron-free gene sequence is called cDNA. RNA DNA Intron Intron Exon Intron Exon Intron 5’ 3’ + RNA Pol Exon CH3 G P Methyl Cap TRANSCRIPTION AAAAA P P Splicing 150 – 250 Poly-A Tail TRANSLATION Translation is the process of protein synthesis, in which genetic information encoded in mRNA is translated into a sequence of amino acids (polypeptide). • Messenger RNA (mRNA) is transported to the ribosomes (in the cytosol) • Ribosomes read the mRNA sequence in triplets of bases called codons • Codons code for specific amino acids according to a genetic code • Amino acids are brought to the ribosome by transfer RNA (tRNA) • Transfer RNA binds to specific codons via complementary anticodons • Anticodons cause tRNA molecules to line up according to codon order • Peptide bonds form between amino acids (catalysed by the ribosome) • Polypeptides are produced as the ribosome moves along the mRNA Hint: MR CAT APP Once the amino acid has been added to a growing polypeptide chain by the ribosome (see diagram below), the tRNA molecule is released to collect a new amino acid. Translation is initiated by a ribosome when it reaches a start codon (AUG) and is terminated when the ribosome reaches a stop codon. The start codon functions to establish the appropriate reading frame in which triplets of bases are read. H H H O N C C OH R MONOMER (AMINO ACID) H H H O H H O N C C N C C R peptide bond R POLYMERISATION (DIPEPTIDE) OH GENETIC CODE The genetic code is the set of rules that identifies which amino acid corresponds to each mRNA codon. It is typically represented as a table. The genetic code possesses two key characteristics: • Universality: Almost every living organism uses the same code (there are a few viral exceptions) • Degeneracy: Some codons code for the same amino acid (there are 64 codons and only 20 amino acids) The Genetic Code UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Phe Leu Leu Ile Met Val UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Pro Thr Ala UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG Translation Tyr STOP His Gln Asn Lys Asp Glu UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG Cys tRNA polypeptide amino acid STOP Trp Arg anticodon Ser Arg Gly codon mRNA ribosome PROTEIN DESTINATIONS Proteins destined to remain within the cytosol (for intracellular use) are synthesised by free ribosomes. Proteins destined for secretion (extracellular use) are synthesised by ribosomes embedded on rough ER. This secretory pathway will involve the transport of proteins within membranous containers (vesicles) that will fuse with the plasma membrane (via exocytosis) to expel the proteins from a cell for extracellular use. The destination of a protein is determined by a short sequence at the start of the polypeptide chain (called the signal recognition peptide). If present, the SRP directs a ribosome to the rough ER (secretory pathway). VESICULAR TRANSPORT Proteins destined for secretion are moved between organelles via a membrane network called the endoplasmic reticulum (ER). The rough endoplasmic reticulum is embedded with ribosomes which synthesise proteins into the lumen of the rough ER. The rough ER then shuttles the proteins to other organelles via vesicles. Proteins destined for secretion are transported to the Golgi apparatus, where they are sorted, stored or modified prior to export. The Golgi complex transports the proteins to the cell membrane via a vesicle, which fuses with the phospholipid bilayer to release the protein externally. The Golgi body can regulate the secretion of proteins from the cell in one of two distinctive ways: • Constitutive: Products are released immediately as they are made • Regulatory: Products are stored for release in response to a signal rough ER vesicle vesicle golgi complex membrane CELL DIFFERENTIATION All cells in a multicellular organism contain the same genetic instructions. The totality of DNA sequences (both genes and non-coding DNA) within a cell or organism is called the genome. However, different genes may be activated in certain tissues, leading to the production of different proteins. The totality of proteins expressed within a cell or organism at a particular time is called the proteome. Because different cell types express different genes and produce different proteins, they may differ in both morphology and function. Cells that are undifferentiated and retain the capacity to form any cell type are known as stem cells. GENE EXPRESSION The expression of genes is coordinated by transcription factors, which are produced by regulatory genes. Transcription factors either mediate or impede the binding of RNA polymerase to the promoter, and hence function to help switch genes on and off. There are two main types of transcription factors: • • Activator proteins bind to enhancer sites and essentially function to increase transcription rates Repressor proteins bind to silencer sites and essentially function to decrease transcription rates The presence of certain transcription factors may be tissue-specific, leading to the differentiation of cells and tissues. Additionally, chemical signals can moderate the activity of transcription factors and hence change gene expression (e.g. hormones may activate target tissues by altering gene expression patterns). The study of changes in organisms as a result of variations in gene expression levels is called epigenetics. Control elements Promoter DNA sequence Normal Expression Up regulation Down regulation RNA polymerase binds promoter Activator binds enhancer site Repressor binds silencer site Gene TRP OPERON An operon is a cluster of genes under the control of a single promoter. The prokaryotic trp operon controls the production of enzymes that are responsible for the synthesis of tryptophan (non-essential amino acid). The expression of the operon is controlled by the trp repressor, which is produced by a regulatory gene. The trp repressor binds to an operator site and inhibits the expression of the operon. Tryptophan acts as a positive inducer molecule by binding to the repressor and activating it. As a consequence of this: • If tryptophan is present, the trp repressor is activated and the operon is off (tryptophan is not made) • If tryptophane is absent, the trp repressor remains inactive and the operon is on (tryptophan is made) The inducer-repressor regulation of the trp operon is an example of a negative feedback loop, whereby the response is the opposite of the stimulus (when levels are low, production of tryptophan increases). Regulatory Gene repressor Promoter Operator trpE trpD trpC trpB trpA Silencer site (represses transcription) tryptophan