Molecular Biology Review Week 9 PDF

Organization of Chromatin Relaxed Chromatin: genes are available for transcription to occur. Closed Chromatin: genes are unavailable; transcription cannot occur. Histone Modification affects gene expression Nucleosomes can slide along DNA. When nucleosomes are spaced closely together, transcription...

Organization of Chromatin Relaxed Chromatin: genes are available for transcription to occur. Closed Chromatin: genes are unavailable; transcription cannot occur. Histone Modification affects gene expression Nucleosomes can slide along DNA. When nucleosomes are spaced closely together, transcription factors can not bind and gene expression is turned off. When the nucleosomes are spaced far apart the DNA is exposed. Transcription factors can bind, allowing gene expression to occur. Modifications to the histones and the DNA affect nucleosome spacing. Methylation of DNA and histones causes nucleosomes to pack tightly together. Transcription factors cannot bind the DNA, and genes are not expressed. Histone acetylation results in loose packing of nucleosomes. Transcription factors can bind the DNA and genes are expressed. Histone Modification: Acetylation Histone acetylation by Histone Acetylation Trabsferase results in relaxed chromatin. Histone deacetylation by Histone Deacytelases results in closed chromatin. Histone Modification: Methylation In general histone methylation results in closed chromatin repressing transcription. But, methylation of some lysine and arginine residues of histones results in transcriptional activation. Gene Expression: In eukaryotes not all genes are transcribed to the same level. The genetic content in each somatic cellin an organism is the same, but not all genes are expressed in every cell. The control of which genes are expressed dictates whether a cell is an eye cell or a liver cell. It is the differential gene expression patterns that arise in different cells that give rise to a complete organism. What are metabolomics? What are transcriptomics? What are proteomics? What are genomics? Talk about Prokaryotic Vs EukaryoticTranscription MRNA stability and degradation The life span of MRNA molecules in the cytoplasm is a key to determining protein synthesis. Eukaryotic mrna is more long lived than prokaryotic mrna. Nucleotide sequences that influence the lifespan of mrna in eukaryotes reside in the untranslated region at the 3rd end of the molecule. Gene organization Humans have 20000 - 25000 protein coding genes. Bacteria are quite varied ranging from 500 - 7500 genes. Compare and contrast mature mrna in prokaryotes and mature mrna in eukaryotes. Mature prokaryotic mrna is polycistronic, while mature eukariotic mrna is monocystronic. Prokariotic gene expression Prokaryotic gene expression is the process of making proteins from DNA in prokaryotic cells, which are cells without a nucleus or organelles. Prokaryotic gene expression can happen simultaneously and in the cytoplasm, unlike in eukaryotic cells, which have separate steps and locations for transcription and translation. Prokaryotic gene expression is also regulated by different mechanisms to control when and how much protein is produced. A cluster of related genes under control of a single promoter is known as a operon. Translation can start before transcription of mrna is complete. Operon Controlled Trp Gene Expression The trp operon is a group of genes that control the production of the amino acid tryptophan in bacteria. The trp operon is turned on when tryptophan levels are low and turned off when they are high. This is done by a protein called the trp repressor that binds to the operator region of the operon and blocks the transcription of the genes. The trp operon is an example of a repressible system and a negative regulation of gene expression The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind the reppresor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan genes . When the tryptophan is absent, the repressor protein does not bind to the operator and the genes are transcribed. Operon Controlled Lac Gene Expression The genes in the lac operon encode proteins that allow the bacteria to use lactose as an energy source. The lac reppresor is a protein that reppreses transcription of the lac operon. It does this by binding to the operator, which partially overlaps with the promoter. When bound the lac reppresor gets in RNA polymerase’s way and keeps it from transcribing the operon. The lac operon is a set of genes in E. coli that are involved in lactose metabolism. It is controlled by two regulators: the lac repressor and the catabolite activator protein (CAP). The lac repressor turns the operon off when lactose is absent, and CAP turns the operon on when glucose is absent. When the catose is not available, the lac reppresor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the lac repressor loses its ability to bind DNA. This change in lac repressor is caused by the small molecule allolactose, an isomer of lactose. Allolactose binds to the lac repressor and makes it change shape so it can no longer bind dna. Allolactose is an example of an inducer, a small molecule that triggers expression of a gene or operon. The lac operon is considered an inducible operon because it is usually turned off, but can be turned on in the presence of the inducer allolactose. Catabolite Activator Protein (CAP) Catabolite activator protein (CAP; also known as cAMP receptor protein, CRP) is a regulatory protein in bacteria that controls the transcription of several genes involved in the metabolism of sugar. CAP binds cAMP, which causes a conformational change that allows it to bind to specific DNA sites in the promoters of the genes it regulates. CAP is a homodimer composed of a ligand-binding domain at the N-terminus and a DNA-binding domain at the C-terminus. When lactose is present, the lac repressor loses its dna binding ability. This clears the way of rna polymerase to bind to the promoter and transcribe the lac operon. But rna polymerase alone does not bind very well to the lac operon promoter. It might take a few transcripts, but it won't do much unless it gets extra help from catabolite activator protein. CAO binds to the region of the DNA just before the lac operon promoter and helps rna polymerase attach to the promoter, driving high levels of transcription. cAMP is a hunger signal made by E. coli when glucose levels are low. cAMP - CAP When glucose levels fall , E. coli may use other sugars for fuel but must transcribe new genes to do so. As glucose supplies become limited, cAMP levels increase. This cAMP binds to the cap protein a positive regulator that binds to an operator region upstream of the genes required to use other sugar sources. Operon Controlled Lac Gene Expression Levels The lac operon will be expressed at high levels if two conditions are met: 1. Glucose must be unavailable, as when this happens, cAMP binds to CAP, making CAP able to bind DNA. Bound CAP helps RNA polymerase attach to the lac operon promoter . 2. Lactose must be available, because when this happens, the lac reppresor will be released from the operator (by binding of allolactose). This allows RNA polymerase to move forward on the DNA and transcribe the operon. These two met conditions allow RNA polymerase to bind strongly to the promoter and give it a clear path for transcription. Transcription of the lac operon is carefully regulated so that its expression only occurs when glucose is limited and lactose is present to serve as an alternative fuel source. Regulation of Eukaryotic Gene Expression Regulation of eukaryotic gene expression is the process that controls when and how much genes produce proteins in eukaryotic cells. It involves many steps and mechanisms, such as transcription, RNA processing, translation, and post-translational modification. It also depends on both cis-acting and trans-acting elements in the DNA. Eukaryotic gene expression is more complex than prokaryotic gene expression because eukaryotes have more genes, more cell types, and more environmental stimuli to respond to. Chromatin accessibility: The structure of chromatin (dna and its organizing proteins) can be regulated. More open or relaxed chromatin makes a gene more available for transcription. Transcription is a key regulatory point for many genes. Sets of transcription factor proteins bind to specific dna sequences in or near a gene and promote or repress its transcription into an rna. Turning a gene off trough dna methylation and histone modification Gene silencing is the process of reducing or preventing the expression of a gene. DNA methylation and histone modification are two types of epigenetic modifications that can affect gene silencing. DNA methylation involves adding a methyl group to the cytosine or adenine bases of DNA, which can interfere with the binding of transcription factors. Histone modification involves adding or removing chemical groups to the histone proteins that wrap around the DNA, which can alter the accessibility of the DNA to the transcription machinery. Non coding types of rna: Small nuclear rna, micro rna, small interfering rna, and heterogeneous nuclear rna. Non coding rna play multiple roles in gene expression. Only a small amount of dna codes for proteins and a very small fraction of the non protein coding dna consists of genes for rna such as rrna and trna. Non coding rnas regulate gene expression at two points: mrna translation and chromatin configuration. The three main types of RNA are: Messenger RNA (mRNA): It carries the genetic information from DNA to the ribosomes for protein synthesis. Ribosomal RNA (rRNA): It forms the core of the ribosomes and catalyzes the formation of peptide bonds between amino acids. Transfer RNA (tRNA): It delivers the specific amino acids to the ribosomes according to the mRNA sequence. Reverse Transcription PCR (RT-PCR) Reverse transcription PCR (RT-PCR) is a technique that combines reverse transcription of RNA into DNA and amplification of specific DNA targets using PCR. It is used to measure the amount of a specific RNA. Quantifying Gene Expression Using PCR Quantifying gene expression using PCR is a technique that measures the amount of a specific gene in a sample. PCR stands for polymerase chain reaction, which is a process that amplifies DNA. There are different types of PCR methods for quantifying gene expression, such as probe-based quantitative PCR (qPCR), relative quantitation, and absolute quantitation. Can convert mrna molecules to cdna in a 1:1 ratio. Can then use real time pcr (qpcr) to count cdna molecules. Can then determine expression levels of mrna used to make cdna. Dna quantification More cycles produces more pcr products. Detection is limited by imager and your eyes. The fewer cycles it takes to detect the pcr product the more copies of the gene you had initially. Quantitative pcr Quantitative PCR (qPCR) is a technique that allows researchers to estimate the quantity of a target DNA sequence in a sample by detecting and measuring the products of a polymerase chain reaction (PCR) as the reaction proceeds.