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Biology 1001A
Tom Haffie

All morphologically complex organisms are eukaryotic - why? Lecture 10: Evolution of Eukaryotes Lecture Outcomes 1. meaning of endosymbiosis, cyanobacteria, lateral gene transfer  endosymbiosis: mitochondria and chloroplast derived from free living prokaryotic cells cyanobacteria: only group of peokaryotes have a photosynthetic structure. lateral gene transfer: organelle genes relocated to the nucleus, puts them under much tighter nuclear control; the function of the gene is retained 2. origin of endomembrane system, nuclear membrane, ER etc.  driven by infolding plasma membrane 3. origin of mitochondria and chloroplasts  Mitochondria is derived from an aerobic bacterium, one of these bacteria that can undergo oxidative phosphorylation. Chloroplasts is derived from cyanobacterium. Aerobic bacterium is taken first. 4. evidence supporting theory of endosymbiosis  Morphology: look like a bacteria Formation/division: the way it diis very much like the way a bacteria divides. Electron transport chains: the only organelles within the eukaryotic cells that have electron transport chains Genome: have their own genomes Transcription/translation machinery: have their own machinery 5. factors driving development of early eukaryotic cells  the ability to use oxygen, oxidative phosphorylation provides huge amounts of ATP, the cell can grow bigger, more proteins can be used, genome is bigger… 6. why eukaryotic cells can be larger and more complex than prokaryotic cells  eukaryotic cells have mitochondria that can produce lots of energy and support an increasing volume 7. evidence for lateral gene transfer from organelles to the nucleus  use southern blot to detect lateral gene transfer between three related species (plant A, B, C); detect the location of oxidase3 gene 8. general idea about how lateral gene transfer is detected (Southern blot)  In a southern blot, run genomic DNA on the gel, DNA single stranded, single strand probe, hybridized, if hybridization occurred, on the membrane you have a sequence similar to your probe. 9. Hypotheses for why genes move to the nucleus from organelles (lateral gene transfer) 10. Possible reasons why certain genes have NOT moved to the nucleus from organelles 11. Role of cpn60 in tracing endosymbiotic and lateral gene transfer event in eukaryotes.  a mitochondria protein, essential for mitochondria to work found in the human nuclear genome showing lateral gene transfer has occurred The evolutionary process of endosymbiosis created organisms with typically "prokaryotic" genes in organelles and typically "eukaryotic" genes in the nucleus. Wow. Lecture 11: Intro to Prokaryotic Gene Structure Lecture Outcomes 1. relative sizes of typical mitochondrial, chloroplast and nuclear genomes  Chlamy cell Mitochondrion 16kb Chloroplast 200kb Nucleus 120,000kb 70 linear chromosomes in the nucleus Circular chromosomes in the mitochondria and chloroplast 2. rubisco structure and assembly from components coded by different genomes  some components of rubisco components are coded by nucleus DNA, some are coded by chloroplast DNA. 3. possible reasons why modern organelle genomes have become dramatically smaller over evolutionary time  Chloroplast and mitochondria lose the gene coding for things like flagella, hexokinase, cell division. (something need in prokaryotes but not in eukaryote)  The genes are been deleted by mutation and deletion. When the organelles have smaller genomes, they are easier to be replicated. That’s a selective advantage.  Lateral gene transfer (gene transferred to the nucleus) 4. possible reasons why genes have moved to the nucleus from organelles over evolutionary time  The nucleus could have more control.  These organelles are involved electron transport. Oxygen metabolism, generate reactive oxygen species, O2+e-=reactive oxygen species, ros is very reactive and very mutagenic creating all kinds of damage in your DNA. Get your DNA out of organelles into the nucleus to get away from ROS.  Nuclear DNA can participate in sexual recombination, while organelle DNA can’t. 5. possible reasons why certain genes have not moved to the nucleus from organelles  Need local control. Need too much time to transfer proteins out of cytosols into the organelle. The gene products are too hard to transport into the organelle.  Genes Doesn’t work in nucleus  Don’t have enough time. Evolution hasn’t stopped. 6. basic mechanism of transcription and translation in prokaryotic organelles vs. eukaryotic nuclear environments  mitochondria and chloroplast have their own gene expression machinery. Transcription and translation happen inside the organelle. Ribosomes are inside the organelle. 7. basic structure and function of RNA polymerase and ribosome  RNA polymerases catalyze the assembly of nucleotides into an RNA strand, rather than the DNA polymerases that catalyze replication. To initiate transcription, RNA polymerase binds to the promoter, unwinds the DNA in that region, and starts synthesizing an RNA molecule at the transcription start point. As RNA polymerase moves along the DNA, unwinding it at the forward end of the enzyme, the new RNA molecule elongates as nucleotides are added one by one The new RNA molecule winds temporarily with the template strand of the DNA into a hybrid RNA–DNA double helix. Beyond this short region of pairing, the growing RNA strand unwinds from the DNA and extends from the RNA polymerase as a single nucleotide chain. As the RNA polymerase passes, the DNA double helix reforms. Elongation of the RNA chain continues until the end of the transcription unit, at which point, RNA synthesis terminates, and the completed RNA transcript and RNA polymerase are released from the DNA  With the help of another protein, RNA polymerase recognizes key DNA sequences in the promoter, binds, and begins transcription of the mRNA. Since all the other types of genes in prokaryotes (for example, tRNA and rRNA genes) have similar promoters, the same RNA polymerase complex can transcribe them all.  In eukaryotes, there are different polymerases for transcribing different types of genes. RNA polymerase II transcribes protein-coding genes. RNA polymerases I and III transcribe genes for non-protein-coding RNAs. A key element of the promoter of most eukaryotic protein-coding genes, the TATA box, is important in transcription initiation. RNA polymerase II itself cannot recognize the promoter sequence. Instead, proteins called transcription factors recognize and bind to the TATA box and then recruit the polymerase. Once the RNA polymerase II–transcription factor complex forms, the polymerase unwinds the DNA and transcription begins.  Ribosome structure A finished ribosome is made up of two parts of dissimilar size, called the large and small ribosomal subunits. Each subunit is made up of a combination of ribosomal RNA (rRNA) and ribosomal proteins. The A site (aminoacyl site) is where the incoming aminoacyl–tRNA (carrying the next amino acid to be added to the polypeptide chain) binds to the mRNA. The P site (peptidyl site) is where the tRNA carrying the growing polypeptide chain is bound. The E site (exit site) is where an exiting tRNA binds as it leaves the ribosome. 8. examples of complementary base pairing in gene expression DNA base pair mRNA, transcription mRNA base pair with itself: once mRNA is produced, it can fold, band, and make base pair on itself; hairpin loop tRNA base pairs with itself: form 3D structure tRNA (anti codon) base pairs with mRNA: translation ribosomal RNA base pairs with itself: 3D structure in order to be catalytic SD box base pair with rRNA in ribosome mRNA base pair with snRNA: during mRNA splicing miRNA/pre-miRNA base pair with itself snRNA base pair with itself (sn: small nuclear) snRNA base pair with either the end of intron DNA contains many types of information. But how is it all "understood" by the cell? Lecture 12: Prokaryotic Gene Function Independent Study Outcomes 1. identify the sequence of standard "start" and "stop" codons  start codon/ initiator codon”: AUG  stop codons/ nonsense or termination codons: UAA, UAG, and UGA 2. identify the function of "start" and "stop" codons  The start codon attracts the first initiator tRNA that codes for methionine and initiates the process of translation.  There is no tRNA binds stop codon. The release factor (protein) always try to get in there, but it is always outcompeted by tRNA. But in this case, there is no competing tRNA. The release factor is able to bind. And translation stops. The release factor is a protein. 3. compare the overall gene expression of prokaryotic vs. eukaryotic cells. Lecture Outcomes 1. relative location of such DNA sequence “signals” as promoter, 5’ and 3’ UTR, “SD box”, start codon, stop codon, transcription terminator etc. 2. mechanism by which each signal is interpreted, or understood, by the cell  promoter attracts attention of RNA polymerase, RNA polymerase interact with DNA and template strand gets transcribed (transcription starts) (but promoter is not transcribed)  terminator sequence in the DNA get transcribed in the mRNA, make a loop structure (pair with itself), and the loop causes destabilize mRNA binds with DNA. mRNA falls off and stops transcribing (transcription ends) (terminator is transcribed, not translated)  start codon of mRNA attracts the first initiator tRNA that codes for methionine and initiates the process of translation. (translation starts) (start codon is transcribed and translated)  SD box is region under DNA, once transcribed into mRNA, the sequence base pair with rRNA, to help the initiation of translation. Not understood by (SD box is transcribed, not translated)  Stop codon can’t bind tRNA, but binds the release factor (a protein, so not base pairing), and translation stops. (Stop codon is transcribed, not translated) 3. relationship between DNA sequence of signals and their function (ie. how would low efficiency promoters be different than high efficiency promoters?  Dimer switch  Promoters have a general common sequence/structure, but they are also quite variable and can drive transcription at different rates. Some promoters are very attractive. The sequence of some promoters is such that polymerase makes a very stable bind and initially transcription very frequently. But other promoters have variable sequences here. They are less and less attractive and less and less efficient at transcription.  A particular terminator stops transcription 60% of the time. Another terminator stops transcription only 40% of the time. Why? Maybe the terminator sequence is longer, makes the bonds in the loop longer and more stable, and therefore termina
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