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AHL Transcription & Translation (7.2-7.3) IB Diploma Biology An SEM micrograph of Ribosomes 7.2.7 Describe the promoter as an example of non-coding DNA with a function. • Only some DNA sequences code for synthesis of polypeptides (single-copy genes) • Non-coding regions (highly-repetitive sequences) have other functions: tRNA production rRNA production (ribosomal RNA) Control gene expression Enhancers: regulatory sequences on DNA which increase the rate of transcription when proteins bind to them. Silencers: sequences on DNA which decrease the rate of transcription when proteins bind to them. 7.2.7 Describe the promoter as an example of non-coding DNA with a function. The Promoter is located near a gene’s location. It is the binding site of RNA polymerase--the enzyme that constructs mRNA from the DNA template during Transcription. Adjacent gene is transcribed while promoter region is not. 7.2.7 Describe the promoter as an example of non-coding DNA with a function. A more complex view of Transcription initiation, including the role of enhancer regions of the DNA sequence… 7.2.1 Gene expression is regulated by proteins that bind to specific base sequences in DNA. • Some proteins are always needed by an organism and so they are constantly being produced… • Other proteins are only needed at certain times or in limited amounts so their production must be controlled… • Gene expression is regulated by environmental factors • Proteins bind to Enhancer sequences to increase transcription of genes for protein synthesis • Proteins bind to Silencer sequences to decrease or inhibit transcription of genes for protein synthesis 7.2.U5 Gene expression is regulated by proteins that bind to specific base sequences in DNA. One well known example of the regulation of gene expression by proteins is the metabolism of lactose in E. Coli bacterium. The diagram below illustrates this example. RNA Polymerase The repressor protein is bound to the operator preventing RNA Polymerase from transcription of the genes DNA Strand Operator is a region of DNA that can regulate transcription, typically inhibiting transcription, such as this silencer sequence. The promoter is a DNA sequence is located near a gene. It acts as the binding site for RNA polymerase. Genes involved in the metabolism (breakdown) of lactose The consequence of the inhibition of the lactose metabolism is that the concentration of undigested lactose now increases in E. Coli … Edited from: http://commons.wikimedia.org/wiki/File:Lac_Operon.svg 7.2.U5 Gene expression is regulated by proteins that bind to specific base sequences in DNA. One well known example of the regulation of gene expression by proteins is the metabolism of lactose in E. Coli bacterium. The diagram below illustrates this example. Lactose binds to the repressor protein inhibiting it: the repressor can no longer bind to the operator. RNA polymerase binds with the promoter, and express the genes (by transcribing them), which in turn synthesizes lactase Lactose molecules build up inside the E. Coli With the synthesis of lactase the lactose is broken down, as it’s concentration decreases the inhibition of the repressor molecules will decrease ‘silencing’ the gene again. 7.2.1 Gene expression is regulated by proteins that bind to specific base sequences in DNA. Example: In E. coli the genes for proteins that digest lactose are silenced unless there is lactose in the cell (lactose binds to the silencer proteins, removing them from the genes so they can be transcribed) 7.2.U5 Gene expression is regulated by proteins that bind to specific base sequences in DNA. Summary of common types of regulating proteins and associated sequences found in eukaryotes. DNA Sequence Binding protein Function Enhancers Activator Activator proteins bind to enhancer sequences of DNA to greatly increase the rate of transcription of a gene. Silencers Repressor Repressor proteins bind to non-coding regions of DNA to either block or reduce the transcription of a gene. Promoter RNA Polymerase A region of DNA located close to a specific gene. Once bound to the sequence RNA polymerase transcribes the gene. 7.2.2 The environment of a cell and of an organism has an impact on gene expression. Epigenetics: the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself… Scientists and philosophers have long debated whether ‘nature’ (genes) or ‘nurture’ (environment) determines the traits and fates of organisms Epigenetics has shown that both play a substantial role as gene expression is clearly impacted by a cell’s environment (ex. human skin cells producing more melanin in high-sun environments… 7.2.2 The environment of a cell and of an organism has an impact on gene expression. In embryonic development, chemicals called morphogens activate gene expression in cells depending on where they are in the embryo to allow for tissue differentiation. Morphogenes regulate the production of transcription factors in a cell. This results in the activation and inhibition of different genes in different cells. This in turn controls how long your fingers should be, where your nose is on your face, and other specifics about body structure. Siamese cats have been selectively-bred for a mutated pigment protein that is only expressed at temperatures below body temperature (thus, these cats only show coloring in their extremities – ears, paws, etc. – where temperatures are lower) 7.2.U6 The environment of a cell and of an organism has an impact on gene expression. The environment of an organism impacts gene expression. For example human hair and skin colour are impacted by the exposure to sunlight and high temperatures. Similarly pigments in the fur of Himalayan rabbits (Oryctolagus cuniculus) are regulated by temperature. Gene C controls fur pigmentation in Himalayan rabbits. The gene is active when environmental temperatures are between 15 and 25°C. At higher temperatures the gene is inactive. In low temperatures Gene C becomes active in the rabbit's colder extremities (ears, nose, and feet) and produces a black pigment. In the warm weather no pigment is produced and the fur is white http://upload.wikimedia.org/wikipedia/commons/0/06/Kr%C3%B3liki_kalifornijskie_666.jpg http://www.alpinecommunitynetwork.com/wp-content/uploads/himalayan-bunny-5-19-11-1_opt4.jpg 7.2.3 Nucleosomes help to regulate transcription in eukaryotes. Eukaryote DNA is associated with histone proteins which wind the DNA to form units called nucleosomes The histone protein tails can be modified: • Acetyl group: neutralizes the positive charge on histones, making DNA less tightly coiled–> increases transcription • Methyl group: maintains positive charge on histones, making DNA tightly coiled –> decreases transcription http://learn.genetics.utah.edu/content/epig enetics/control/ 7.2.U2 Nucleosomes help to regulate transcription in eukaryotes. Methylation is the addition of methyl groups to DNA Methylation of DNA inhibits transcription *Chromatin is a complex of DNA, protein and RNA. Tightly packed chromatin which cannot be transcribed is referred to as heterochromatin. Processes that inhibit transcription bind the DNA more tightly to the histone making it less accessible to transcription factors (forming heterochromatin). Edited from: http://www.nature.com/neuro/journal/v13/n4/images/nn0410-405-F1.jpg 7.2.U2 Nucleosomes help to regulate transcription in eukaryotes. Acetylation is the addition of Acetyl groups to histones Acetylation promotes transcription n.b. Methylation of histones can also occur, this process can both promote and inhibit transcription. Processes that promote transcription bind the DNA more loosely to the histone making it more accessible to transcription factors (forming euchromatin*). *Chromatin is a complex of DNA, protein and RNA. Loosely packed chromatin which can be transcribed is referred to as euchromatin. Edited from: http://www.nature.com/neuro/journal/v13/n4/images/nn0410-405-F1.jpg 7.2.3 Nucleosomes help to regulate transcription in eukaryotes. 7.2.U2 Nucleosomes help to regulate transcription in eukaryotes. Changes in the environment affect the cell metabolism, this in turn can directly or indirectly affect processes such as Acetylation & Methylation. Methylation and acetylation mark the DNA to affect transcription. These these markers are known as epigenetic tags*. For a new organism to grow it needs unmarked DNA that can develop into lots of different specialised cell types. Reprogramming scours the genome and erases the epigenetic tags to return the cells to a genetic "blank slate”. *The branch of genetics concerned with hertible changes not caused by DNA is called Epigenetics. For a small number of genes, epigenetic tags make it through this process unchanged hence get passed from parent to offspring. http://learn.genetics.utah.edu/content/epigenetics/inheritance/images/Reprogramming.jpg 7.2.8 Analyze changes in DNA methylation patterns. Direct methylation of DNA (not to histone tails) is thought to affect gene expression. • Increased methylation of DNA decreases gene expression • DNA methylation is variable during our lifetime • Amount of methylation depends on environmental factors, like diet • Evidence for heritability of methylated DNA • EX: Diet of pregnant mice has been shown to influence weight and fur color of offspring (left) 7.2.4 Transcription occurs in a 5’ to 3’ direction. 5’ 3’ 7.2.5 Eukaryotic cells modify mRNA after transcription. Introns are Interruptions within the coding sequences of a gene transcript (mRNA) Exons are the Expressed sections of the gene transcript (mRNA) that are translated into polypeptides http://bcs.whfreeman.com/thelifewire/co ntent/chp14/1401s.swf 7.2.5 Eukaryotic cells modify mRNA after transcription. In addition to splicing out the Introns from the pre-mRNA, a 5’ cap is added to the mRNA transcript and a Poly-A tail is added to the 3’ end to protect against degradation of the coding sections of the mRNA (similar to telomeres in DNA) 7.2.6 Splicing of mRNA increases the number of different proteins an organism can produce. • The Proteome (set of proteins) of an organism is actually much larger than its Genome (set of genes) • The main way there can be more proteins than there are genes is due to Alternative Splicing: • • • • Proteins are often translated from mRNA with multiple exons. The exons can be spliced together differently, result in in a different sequence of amino acids. Consequently, a number of different protein structures and functions are possible from the same mRNA 1 fruit fly gene can produce 38,000 different mRNAs / proteins based on the different ways it’s exons can be spliced together! 7.2.6 Splicing of mRNA increases the number of different proteins an organism can produce. 7.3.12 Use molecular visualization software to analyze the structure of eukaryotic ribosomes and a tRNA molecule. Ribosome Structure: • Made up of proteins and ribosomal RNA (rRNA) • Large subunit (50S) & small subunit (30S) – make up 80S ribosome • 3 binding sites for tRNA (A site, P site, E site) • tRNA enters A site, shifts to P site, and exits E site • 2 tRNAs can bind to the surface of the ribosome at a time, 1 mRNA can bind to surface of small subunit 7.3.12 Use molecular visualization software to analyze the structure of eukaryotic ribosomes and a tRNA molecule. tRNA Structure: • Double stranded sections by complementary base pairing • Anticodon of 3 bases in a 7-base loop • 2 other loops • 3’ end has amino acid binding site with CCA sequence of unpaired bases 7.3.11 tRNA-activating enzymes illustrate enzyme-substrate specificity and the role of phosphorylation. 7.3.11 tRNA-activating enzymes illustrate enzyme-substrate specificity and the role of phosphorylation. http://www.phschool.com/science/biology _place/biocoach/translation/addaa.html http://highered.mheducation.com/sites/9834092339/stud ent_view0/chapter15/aminoacyl_trna_synthetase.html 7.3.1 Initiation of translation involves assembly of the components that carry out the process P-site of ribosome A-site of ribosome 7.3.2 Synthesis of the polypeptide involves a repeated cycle of events. Elongation: A series of repeated steps… • • • • • Ribosome moves 3 bases (one codon) along the mRNA (5’ -> 3’) tRNA at P-site moves to E-site, allowing it to disengage tRNA complementary to the codon at A-site enters Peptide bond forms between AA’s in A and P sites Process continues many times 7.3.3 Disassembly of the components follows termination of translation 7.3.3 Disassembly of the components follows termination of translation http://www.stolaf.edu/people/giannini/flashanimat/molgen etics/translation.swf http://highered.mheducation.com/sites/0072507470/st udent_view0/chapter3/animation__how_translation_w orks.html 7.3.4 Free ribosomes synthesize proteins for use primarily within the cell / 7.3.5 Bound ribosomes synthesize proteins primarily for secretion or for use in lysosomes. Location of protein synthesis: cell functions & protein synthesis are compartmentalized (by organelles) • Proteins that will be used by the cell in cytoplasm, mitochondria, and chloroplasts are synthesized on free ribosomes in the cytoplasm • Proteins that will be secreted or used by lysosomes are synthesized on bound ribosomes found on the RER 7.3.4 Free ribosomes synthesize proteins for use primarily within the cell / 7.3.5 Bound ribosomes synthesize proteins primarily for secretion or for use in lysosomes. 7.3.13 Identify polysomes in an electron micrograph. • Polysome: Many ribosomes simultaneously translating the same mRNA (allows for faster protein synthesis) • Polysomes appear as beads on a string in electron micrographs • “Beads” represent multiple ribosomes attached to a single mRNA molecule • Poly = many, some = ribosome 7.3.6 Translation can occur immediately after transcription in prokaryotes due to the absence of a nuclear membrane. In prokaryotes, since there is no nucleus, transcription and translation can be directly coupled (see below). Ribosomes attach to the mRNA as it is being synthesized from the DNA template. http://www.stolaf.edu/people/giannini/flashan imat/proteins/protein%20structure.swf