5 - Gene Expression.pdf

BIOCHEMISTRY: BUCM 2024-2025 MELVIN G. BERIN, MD References: 1. Harper’s Illustrated Biochemistry 2. Textbook of Biochemistry - For Medical Students, 6th Ed by Vasudevan, Sreekumari & Vaidyanathan Gene Expression multistep process that ultimately results in the production o...

BIOCHEMISTRY: BUCM 2024-2025 MELVIN G. BERIN, MD References: 1. Harper’s Illustrated Biochemistry 2. Textbook of Biochemistry - For Medical Students, 6th Ed by Vasudevan, Sreekumari & Vaidyanathan Gene Expression multistep process that ultimately results in the production of a functional gene product, either ribonucleic acid (RNA) or protein Ability of a cell to control or regulate what products (RNA or protein) it makes from its DNA CENTRAL DOGMA OF MOLECULAR BIOLOGY 2 Main Groups of Genes 1. Structural Genes Protein Coding Genes About 20,400 in the human body 2% of human DNA 2. Regulatory Genes Genes that regulate the proteins that are produced by the structural genes 98% of human DNA REGULATION OF PROKARYOTIC GENE EXPRESSION Messenger RNA transcription from bacterial operons An operon is a cluster of genes that are transcribed together to give a single messenger RNA (mRNA) molecule, which therefore encodes multiple proteins Lactose operons contain an operator, a segment of DNA that regulates the activity of the structural genes of the operon by reversibly binding a protein known as the repressor. If the operator is not bound by the repressor, RNA polymerase (RNA pol) binds the promoter, passes over the operator, and reaches the protein- coding genes that it transcribes to mRNA. If the repressor is bound to the operator, the polymerase is blocked and does not produce mRNA. As long as the repressor is bound to the operator, no mRNA (and, therefore, no proteins) are made. However, when an inducer molecule is present, it binds to the repressor, causing the repressor to change shape so that it no longer binds the operator. When this happens, RNA pol can initiate transcription. The lac operon contains the genes that code for three proteins involved in the catabolism of the disaccharide lactose: o the lacZ gene codes for β-galactosidase, which hydrolyzes lactose to galactose and glucose; o the lacY gene codes for a permease, which facilitates the movement of lactose into the cell; o the lacA gene codes for thiogalactosidetransacetylase, which acetylates lactose. (Note: The physiologic function of this acetylation is unknown.) All of these proteins are maximally produced only when lactose is available to the cell and glucose is not. 1. When only glucose is available: In this case, the lac operon is repressed (turned off). Binding of the repressor interferes with the binding of RNA pol to the promoter, thereby inhibiting transcription of the structural genes. This is an example of negative regulation 2. When only lactose is available: In this case, the lac operon is induced (maximally expressed, or turned on). A small amount of lactose is converted to an isomer, allolactose. This compound is an inducer that binds to the repressor protein, changing its conformation so that it can no longer bind to the O site. In the absence of glucose, adenylyl cyclase is active, and cAMP is made and binds to the CAP. The cAMP–CAP trans-acting complex binds to the CAP site, causing RNA pol to initiate transcription with high efficiency at the promoter site. This is an example of positive regulation. The transcript is a single polycistronic mRNA molecule that contains three sets of start and stop codons. Translation of the mRNA produces the three proteins that allow lactose to be used for energy production by the cell. (Note: In contrast to the inducible lacZ, lacY, and lacA genes, whose expression is regulated, the lacI gene is constitutive. Its gene product, the repressor protein, is always made and is active unless the inducer is present.) 3. When both glucose and lactose are available: In this case, the lac operon is uninduced, and transcription is negligible, even if lactose is present at a high concentration. Adenylyl cyclase is inhibited in the presence of glucose (a process known as catabolite repression) so no cAMP–CAP complex forms, and the CAP site remains empty. Therefore, the RNA pol is unable to effectively initiate transcription, even though the repressor is not bound to the O site. Consequently, the three structural genes of the operon are expressed  only at a very low (basal) level. C. Tryptophan Omepron includes five genes that encode enzymes needed for tryptophan biosynthesis, along with a promoter (RNA polymerase binding site) & an operator (binding site for a repressor protein). The genes of the trp operon are transcribed as a single mRNA. The trp operon is regulated by the trp repressor or attenuation Trp Repressor o The trp repressor binds and blocks transcription only when tryptophan is present. When tryptophan is around, it attaches to the repressor molecules and changes their shape so they become active. o Trytophan, which switches a repressor into its active state, is called a corepressor. More trp operon regulation: Attenuation o Like regulation by the trp repressor, attenuation is a mechanism for reducing expression of the trp operon when levels of tryptophan are high. However, rather than blocking initiation of transcription, attenuation prevents completion of transcription. o When levels of tryptophan are high, attenuation causes RNA polymerase to stop prematurely when it's transcribing the trp operon. Only a short, stubby mRNA is made, one that does not encode any tryptophan biosynthesis enzymes. Attenuation works through a mechanism that depends on coupling (the translation of an mRNA that is still in the process of being transcribed). The Leader The section between the operator and the first gene of the operon and is called the leader. The leader encodes a short polypeptide and also contains an attenuator sequence. The attenuator does not encode a polypeptide, but when transcribed into mRNA, it has self-complementary sections and can form various hairpin structures. Once RNA polymerase has started transcribing the operon, a ribosome can attach to the still- forming transcript and begin translating the leader region. The polypeptide encoded by the leader is short, just 14 amino acids long, and it includes two tryptophan (Trp) residues. The tryptophans are important because:  If there are plenty of tryptophan, the ribosome won't have to wait long for a tryptophan- carrying tRNA, and will rapidly finish the leader polypeptide. If there is little tryptophan, the ribosome will stall at the Trp codons (waiting for a Trp-carrying tRNA) and will be slow to finish translation of the leader. Why does it matter if the ribosome translates the leader quickly or slowly? As mentioned above, the leader is followed by an attenuator region, which (in its mRNA form) can stick to itself to form different hairpin structures. One structure includes a transcription termination signal, while the other does not end termination (and in fact, prevents formation of the terminator hairpin) If the ribosome translates slowly, it will pause, and its pausing causes formation of the antiterminator (non-terminating hairpin). This hairpin prevents formation of the terminator and allows transcription to continue. Low Trp levels: When there is not much tryptophan available in the cell, the ribosome will stall at the Trp codons while translating the short polypeptide at the start of the leader. This stalling causes regions 2 and 3 to associate with one another in a hairpin. This hairpin, called an antiterminator hairpin, prevents the terminator hairpin (regions 3 and 4 paired up) from forming. Termination does not occur and RNA polymerase continues transcribing, producing a transcript that includes the trpE- trpA genes. High Trp levels: The ribosome does not stall at the Trp codons while synthesizing the leader polypeptide, because Trp is abundant (and there are thus plenty of Trp-carrying tRNAs around). Instead, the ribosome quickly synthesizes the leader polypeptide, reaches the stop codon, and detaches from the mRNA. This  leaves regions 1 and 2 free to pair up, at which point regions 3 and 4 will also pair up and form a terminator hairpin. The terminator hairpin causes RNA polymerase to detach from the DNA and from the transcript, ending termination. A short mRNA consisting of the leader region is all that gets produced; the trpE-trpA genes are never transcribed. The logic of attenuation is the same as that of regulation by the trp repressor. In both cases, high levels of tryptophan in the cell shut down the expression of the operon. This makes sense, since high levels of tryptophan mean that the cell does not need to make more biosynthetic enzymes to produce additional tryptophan. Note: Transcriptional attenuation can occur in prokaryotes because translation of an mRNA begins before its synthesis is complete. This does not occur in eukaryotes because the presence of a membrane-bound nucleus spatially and temporally separates transcription and translation. REGULATION OF EUKARYOTIC GENE EXPRESSION In eukaryotes like humans, gene expression involves many steps, and gene regulation can occur at any of these steps. However, many genes are regulated primarily at the level of transcription. Components of Eukaryotic gene 1. Exons o The parts of the gene sequence that are expressed o DNA sequence that encode a polypeptide 2. Introns - o the parts of the gene sequence that are not expressed in the protein o they come in between the exons 3. Start site – o where RNA Polymerase II binds o RNA Polymerase II is a complex of 12 proteins that synthesizes the mRNA 4. Promoter a. Core Promoter o DNA sequence located about 40 bases upstream of the start site. o Found in ALL eukaryotic genes o most common is the TATA promoter o about 50 proteins can bind the Core promoter for gene expression complexes. These complexes include i. Trancription Factor II D ▪ Complex consist of the TATA Binding Protein which recognizes and binds to the TATA Box ii. Transcription Factor II B ▪ This protein helps the TATA Binding Protein interact with RNA polymerase II b. Proximal (Upstream) Promoter o Segments of DNA found upstream of the Core promoter o They can also bind with activators and repressors needed to regulate gene expression  o Unlike Core promoter, upstream promoters vary from gene to gene 5. Enhancer/ Silencer o DNA sequence that are from away from the gene o Maybe upstream or downstream o Bind special transcription factor proteins that increase the rate of transcription The TATA-box is the site of preinitiation complex formation, which is the first step in transcription initiation in eukaryotes. Formation of the preinitiation complex begins when the multi-subunit transcription factor (TFIID) binds to the TATA box. It then allows other transcription factors and eventually RNA Polymerase II to bind. Transcription Factors are proteins involved in the process of converting or transcribing DNA into RNA. Transcription factors include a wide number of proteins, excluding RNA polymerase, that initiate and regulate the transcription of genes. Main function is to help turn specific genes "on" or "off" by binding to nearby DNA Structure: All transcription factors have two domains that are necessary for their function. The first is a DNA binding domain (DBD). DBDs attach to specific DNA sequences that are upstream to a regulated gene. This specific region is called a promoter or response element. The transactivation domain (TAD) is where other proteins (co-regulatory proteins) bind to the transcription factor. A third element is sometimes present and called a signal-sensing domain (SSD). This region, when present, allows signaling molecules to bind the transcription factor. Clinical Correlates Huntington’s Disease (HD) An autosomal dominant neurodegenerative disorder caused by mutations in the HTT gene, which encodes the huntingtin protein. The mutation involves an expansion of CAG trinucleotide repeats in the HTT gene, leading to an abnormal version of the huntingtin protein. This protein accumulates in neurons, causing cell death. Manifests with motor dysfunction, cognitive decline, and psychiatric symptoms. Gene expression studies have revealed that mutant huntingtin protein affects transcriptional regulation and protein-protein interactions, contributing to disease pathology. Research is ongoing to develop gene silencing therapies to reduce the expression of the mutated gene. Duchenne Muscular Dystrophy (DMD) An X-linked recessive disorder characterized by progressive muscle degeneration and weakness. It is caused by mutations in the DMD gene, which encodes dystrophin, a protein crucial for muscle fiber integrity. Mutations, such as deletions or nonsense mutations in the DMD gene, lead to the absence or abnormal expression of dystrophin, resulting in muscle fiber damage and necrosis. Patients with DMD exhibit delayed motor milestones, muscle weakness, and cardiopulmonary complications. Understanding the gene expression of dystrophin has enabled the development of therapies like exon skipping, which aims to restore the reading frame of the DMD gene and produce a functional, albeit shorter, dystrophin protein. Copolymer-based muscle membrane stabilization of dystrophic muscle. a Representation of intact muscle membrane with dystrophin anchoring the DGC to the actin cytoskeleton. b Membrane instability caused by the lack of dystrophin leads to pathological increases in intracellular Ca 2+ concentration. c Copolymer stabilization of the damaged membrane via insertion of its hydrophobic PPO block (red) prevents entry of extracellular Ca 2+ into the cell