Unit Two: Molecular Genetics. 5.5 Control Mechanisms 5.7 Key Differences 5.8 Genes and Chromosomes

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1 Unit Two: Molecular Genetics 5.5 Control Mechanisms 5.7 Key Differences 5.8 Genes and Chromosomes

2 Control Mechanisms Not all genes need to be produced at all times. Example: alcohol dehydrogenase Methods to control transcription and translation, depending on need. Housekeeping genes always on Transcription factors turn genes on Four levels of gene regulation: Transcriptional, post-transcriptional, translational, post-translational Look at two control mechanisms in prokaryotic cells.

3 How can genes be regulated? Transcription factors turn genes on There are four levels of control: Transcriptional which genes are transcribed Posttranscriptional Which is??? Translational How often transcripts are translated, how long the mrna transcript is active before being destroyed by cytoplasmic enzymes. Posttranslational before protein becomes functional, and remains functional by adding chemical groups.

4 lac Operon Lactose disaccharide from milk products Consists of glucose and galactose E.coli can use lactose as energy source must first split into monomers. Enzyme - -galactosidase E. coli only produce when lactose is in enviro Use negative regulation blocks production if lactose is not present

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6 E. coli, lac operon Three enzymes are used to take up and metabolize lactose including β- galactosidase, clustered together in the lac operon Regulatory gene laci located outside operon, codes for allosteric repressor that can switch lac operon off by binding to the operator. Repressor is active by itself turning lac operon off. Inducer molecule is necessary to inactivate the repressor.

7 -galactosidase operon cluster of genes under control of one promoter, one operator Operons only used in prokaryotes to regulate. Operons do not code for protein only regulatory sequences lac operon three genes that code for proteins in metabolism of lactose. Genes lacz - -galactosidase, lacy - -galactosidase permease causes lactose to permeate the cell membrane, and laca transacetylase unknown function

8 Inducer Inducer for lac operon allolactose isomer of lactose formed in small amounts when lactose is present in cell. No lactose = No allolactose = lac repressor active = lac operon silent (no protein) Lactose = allolactose - binds to repressor = no repressor, lac operon active (proteins made) lac operon produces on demand mrna for enzymes of the lactose pathway.

9 operator promoter starting point for transcription transcription termination RNA polymerase cannot bind to promoter lac repressor (active) transcription blocked allolactose binding site inactivated repressor transcription begins allolactose (inducer)

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11 trp operon trp operon regulates the production of the amino acid tryptophan E. coli capable of producing tryptophan, but if trp is available in enviro no need. trp operon regulation repressed when tryptophan levels are high trp operon consists of 5 genes 3 code for polypeptides enzymes needed for tryptophan synthesis.

12 trp operon tryptophan metabolism Repressible operon for synthesis of amino acid tryptophan pathway. When end product of pathway (tryptophan) is present it represses the gene production. Operon includes promoter and operator region. Regulatory gene is located outside the operon, synthesized in inactive form, and remains unless tryptophan is present.

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14 1. DNA packing How do you fit all that DNA into nucleus? DNA coiling & folding double helix nucleosomes chromatin fiber looped domains chromosome from DNA double helix to condensed chromosome

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17 Organization of DNA DNA itself is 2 nm in diameter 200 bp of DNA are wrapped around a histone core to make a nucleosome. Each nucleosome is 10 nm in diameter. Nucleosomes resemble beads on a string. The next level of packaging is a 30 nm fiber, this is the form of chromatin. There is further compacting creating a chromatid that is 700 nm in diameter.

18 Eukaryotic Gene Expression A typical human cell most likely expresses about 20% of its genes at any one time. Highly differentiated cells, express even fewer of their genes. Almost all cells in an organism contain an identical genome, but the subset of genes expressed in the cells of each type is unique. These differences are what allows cells to carry out specific functions differential gene expression.

19 Eukaryotic Gene Control Mechanisms Eukaryotic gene expression is more complex due to there being a greater number of steps. Four levels of control: Transcriptional (as mrna is being synthesized) Post-transcriptional (as mrna is being processed) Translational (as the protein is being synthesized) Post-translational (after the protein has been synthesized)

20 Transcriptional Regulation The most common type of regulation is transcriptional. Gene promoters are not accessible to the proteins that initiate transcription when DNA is wrapped around histone proteins. For a gene to be transcribed it must be partially unwound to expose the promoter.

21 In one type of transcriptional regulation an activator molecule binds to a sequence upstream of the gene s promoter and signals remodelling of chromatin. The histone s core proteins are displaced from the DNA and the promoter is exposed.

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23 In histone acetylation, acetyl groups are attached to histones. This loosens the histone s association with DNA, allowing the promoter to become accessible. As well transcription can be regulated by activators and repressors that increase or decrease the rate of transcription, by binding to the promoter along with the general transcription factors.

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25 Methylation is another method of gene regulation. A methyl group is added to the cytosine bases in the promoter of a gene, and it inhibits transcription. This is used to silence genes, such as those for hemoglobin in all cells, except red blood cells.

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27 Post-transcriptional regulation One type is alternative splicing, which produces different mrnas from pre-mrna by removing different combinations of introns. Depending on which protein is required by the cell, an intron in one pre-mrna may be considered an exon in another pre-mrna. This allows for the production of a family of related polypeptides.

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29 Masking proteins bind to mrna, and prevent protein synthesis. Later other proteins remove these masking proteins to allow the mrna to be translated. This type of regulation is found in animal eggs as a way to keep mrnas inactive until the egg has been fertilized.

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31 Degradation of mrnas acts as another control mechanism. A regulatory molecule, such as a hormone will directly, or indirectly affect the rate of mrna breakdown. Example: in the mammary gland of a rat, the half-life of the mrna for casein (a milk protein) is 5 h, but in the presence of the hormone prolactin, the half-life becomes 92 h. Prolactin signals the need for milk, increasing the rate of translation of the protein casein.

32 Translational regulation involves changes in the length of the poly(a) tail on the mrna molecule. If the tail is too short, it will not be translated.

33 Post-translational regulation After translation and the proteins have been synthesized, the cell can still regulate the availability of functional proteins. Three methods are used: Processing Chemical modification Degradation

34 When proteins are first made, they are in an inactive form, and must be activated by various processing mechanisms. For example insulin is initially synthesized as pro-insulin, an inactivated precursor. The initial protein must be split in order to become an active hormone.

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36 Many proteins undergo chemical modifications that make them functional. Regulatory proteins are commonly activated or inactivated by the reversible addition of phosphate groups. Proteins that are found on the surface of animal cells add on sugars. Cell-surface proteins and many other must also be transported to the target destination in order to function.

37 Degradation Proteins are subject to constant degradation both inside and outside the cell. Some proteins are only used for a few minutes before they are broken down, others can last the entire lifespan of an organism. Short-lived proteins are tagged with a small protein called ubiquitin, which is then recognized by proteasomes that then unfold the protein, and break it into small peptides.

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39 Key Differences between: Genome Transcription Translation Prokaryotes Double stranded Circular All region code for polypeptides except promoter Operons present In cytoplasm, coupled with translation, no splicing Start: formylmethionine Recognize Shine- Dalgarno sequence on mrna Ribosomes smaller Eukaryotes Double stranded Linear - chromosomes arrangement Coding and non-coding No operons In nucleus, Spliceosomes join exons Start: methionine Recognize 5 cap on mrna Ribosomes larger

40 Prokaryotes Eukaryotes Mitochondria of Eukaryotic cells Chloroplasts of Photosynthetic eukaryotes DNA Replication 1 single circular chromosom e Binary fission Multiple linear in a nucleus Mitosis 1 single circular chromosome Binary fission Ribosomes 70 S 80 S 70 S 70 S Electron transport chain Size (approx) Found in plasma membrane around cell ~1-10 microns Found only in mitochondri a and chloroplasts ~ microns Found in plasma membrane around mitochondri a ~1-10 microns 1 single, circular chromosome Binary fission Found in plasma membrane around chloroplasts ~1-10 microns Appearance ~3.2 - ~3.8 ~1.5 billion ~1.5 billion ~1.5 billion

41 Come up with a hypothesis Endosymbiotic theory hypothesis originally proposed that: mitochondria are the result of endocytosis of aerobic bacteria chloroplasts are the result of endocytosis of photosynthetic bacteria in both cases by large anaerobic bacteria who would not otherwise be able to exist in an aerobic environment. this arrangement became a mutually beneficial relationship for both cells (symbiotic).

42 Endosymbiotic theory original hypothesis proposed that aerobic bacteria (that require oxygen) were ingested by anaerobic bacteria (poisoned by oxygen), and may each have had a survival advantage as long as they continued their partnership creating a double membrane bound organelle Supported by: fossil evidence timeline Organelles have own DNA, replicate independently Mitochondrial DNA only from maternal

43 By the numbers The human genome contains 3.2 billion base pairs (A, C, T, and G). The average gene consists of 3,000 base pairs, but sizes vary greatly, with the largest known human gene being dystrophin at 2.4 million base pairs. The total number of genes is estimated at 25,000, much lower than previous estimates of 80,000 to 140,000 that had been based on extrapolations from gene-rich areas as opposed to a composite of gene-rich and gene-poor areas. The human genome sequence is almost exactly the same (99.9%) in all people. Functions are unknown for more than 50% of discovered genes.

44 About 2% of the genome encodes instructions for the synthesis of proteins. Repeat sequences that do not code for proteins make up at least 50% of the human genome. Repeat sequences are thought to have no direct functions, but they shed light on chromosome structure and dynamics. Over time, these repeats reshape the genome by rearranging it, thereby creating entirely new genes or modifying and reshuffling existing genes. During the past 50 million years, a dramatic decrease seems to have occurred in the rate of accumulation of repeats in the human genome.

45 Particular gene sequences have been associated with numerous diseases and disorders, including breast cancer, muscle disease, deafness, and blindness. Stretches of up to 30,000 C and G bases repeating over and over often occur adjacent to gene-rich areas, forming a barrier between the genes and the "junk DNA." These CpG islands are believed to help regulate gene activity. Chromosome 1 (the largest human chromosome) has the most genes (3,168), and Y chromosome has the fewest (344).

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48 How It's Arranged The human genome's gene-dense "urban centers" are predominantly composed of the DNA building blocks G and C. In contrast, the gene-poor "deserts" are rich in the DNA building blocks A and T. GC- and AT-rich regions usually can be seen through a microscope as light and dark bands on chromosomes. Genes appear to be concentrated in random areas along the genome, with vast expanses of noncoding DNA between.

49 The human genome has a much greater portion (50%) of repeat sequences than the mustard weed (11%), the worm (7%), and the fly (3%). Over 40% of predicted human proteins share similarity with fruit-fly or worm proteins. Although humans appear to have stopped accumulating repeated DNA over 50 million years ago, there seems to be no such decline in rodents.

50 This may account for some of the fundamental differences between hominids and rodents, although gene estimates are similar in these species Scientists have proposed many theories to explain evolutionary contrasts between humans and other organisms, including those of life span, litter sizes, inbreeding, and genetic drift.

51 Variations and Mutations Scientists have identified millions of locations where single-base DNA differences occur in humans. This information promises to revolutionize the processes of finding DNA sequences associated with such common diseases as cardiovascular disease, diabetes, arthritis, and cancers. The ratio of germline (sperm or egg cell) mutations is 2:1 in males vs females. Researchers point to several reasons for the higher mutation rate in the male germline, including the greater number of cell divisions required for sperm formation than for eggs.

52 Applications The draft sequence already is having an impact on finding genes associated with disease. Over 30 genes have been pinpointed and associated with breast cancer, muscle disease, deafness, and blindness. Additionally, finding the DNA sequences underlying such common diseases as cardiovascular disease, diabetes, arthritis, and cancers is being aided by the human variation maps (SNPs) generated in the HGP in cooperation with the private sector. These genes and SNPs provide focused targets for the development of effective new therapies.

53 Future Challenges One of the greatest impacts of having the sequence may well be in enabling an entirely new approach to biological research. In the past, researchers studied one or a few genes at a time. With wholegenome sequences and new high-throughput technologies, they can approach questions systematically and on a grand scale. They can study all the genes in a genome, for example, or all the transcripts in a particular tissue or organ or tumor, or how tens of thousands of genes and proteins work together in interconnected networks to orchestrate the chemistry of life.

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