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unit 1 dna and rna.doc

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Kathleen Gilmour

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Unit 1: DNA and RNA- Structure and Function What is the structure of DNA and how do we know? The Basic Building Blocks for DNA and RNA -4 nitrogenous bases (adenine, cytosine, guanine, thymine) -purines = adenine and guanine -pyrimidine = cytosine and thymine -nucleoside = base and pentose sugar (e.g.: adenosine, cytidine, guanosine, thymidine / uridine) -if the sugar is a deoxyribose = deoxyribonucleoside -e.g.: deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine -nucleotide = nucleoside and phosphate -the differences between RNA and DNA are: -in RNA, uracil replaces thymine (uracil has no Me group) -ribose replaces deoxyribose (ribose has a -OH group on the 2') Archetypal Structural Forms for DNA and RNA -baceterium has circular dsDNA (double stranded) organized into a protein/DNA complex called the nucleoid -circular DNA are more easily replicated -eukaryotes have linear dsDNA organized into protein/DNA-containing chromosome -mitochondrian and chloroplast have circular dsDNA that are found in protein/DNA complexes called nucleoid -mitochondria and chloroplast evolved from bacterium -viruses can come as circular/linear, ss/ds, DNA/RNA in protein/DNA complexes called capsids or viral particles/phage particles which bind DNA/RNA into DNA/RNA-protein complexes -this does not apply to prions (a protein that is infectious but is encoded by a genome; acts as an aberrant variant of a protein) -when they interact with the normal protein, they turn it into the aberrant form which leads to brain damage -viroids only infect plants -made up of a small, circular, ssRNA made up of a few hundred nucleotides -have no protein -reasons why we need the proteins in the complexes: -condensation of the DNA molecule occurs by wrapping caused by proteins -proteins interact with the DNA to neutralize the negative charge caused by the phosphate groups Is DNA always the genetic material? -DNA is the genetic material Transformation Experiments -involves changing one phenotype of a cell into another 1) Griffith's Experiments -first experiment to show transformation -Griffith's identified 2 types of Streptococcus pneumoniae (diplococcus) -1 is the smooth form (S form) that has a capsule around the outside; these replicate and cause death -1 is the rough form (R form) has no capsule and does not cause death -if the virulent S-form is heat treated and killed (so that the cell is dead but the chemicals inside are not dead) and injected into the mice, they survive -if the heat treated S-form is mixed with the safe R-form and injected into the mouse, the mouse died -the living cells which were not virulent were converted to virulent cells -if the mixture of cells were recovered and propagated, they are still virulent (the change was permanent and can be inherited now = transformation) Caveats: -the transformation was genetic and was inherited -there are many mechanisms for transferring DNA between cells which are not unique (i.e.: transformation, transduction, conjugation) -DNA is everywhere; when we die we release DNA (carbon, nitrogen, phosphate) and other organisms take it up 2) Avery-MacLeod-McCarty Experiment -1940s -attempted to define the transforming principle by treating the transforming principle with enzymes (not pure) and see if transformation was still present -used bacteria particles; not virus particles -take the extract from the other experiment and treated it with something and saw if it survived treatment -if you treat it with something and it can no longer transform, that thing must be doing something bad to the transforming principle -no treatment = transformation -treatment with trypsin (a protease) = transformation -treatment with chymotrypsin (a protease) = transformation -treatment with ribonuclease (a RNAase) = transformation -treatment with deoxyribonuclease (a DNAase) = NO transformation; therefore DNA was the transforming principle Caveats: -experiment had no controls; extracts (i.e.: deoxyribonuclease, ribonuclease, etc.) were not pure -if there was control, you would need to demonstrate that a deoxyribonuclease only digested DNA and nothing else 3) Chargaff's Experiments -was interested in the chemical nature of DNA -supplied the information that Watson and Crick used to develop the model of DNA -Chargaff tried to take different parts of DNA (from different organisms), degrade it chemically and look to see how much DNA was in those 4 forms -in double stranded DNA, C and G / A and T had the same value within experimental error -sometimes this rule did not apply: -if there was RNA contaminating it, uracil would have replaced thymine -in RNA, U is not equal to A because RNA is single stranded -if RNA was double stranded, A = U and G = C -did not apply in single stranded DNA/RNA e.g.: A and T may not be the same in single stranded DNA -some bases are modified in organisms; there are more than 4 types of nitrogenous bases 4) Hershey-Chase Experiment -use of radio-isotopes to label specific sub-cellular fractions 35 - S32se to label proteins - P use to label DNA -took advantages of bacteriophages -the protein is labelled with S35 (i.e.: the amino acids, cysteine and methionine) -P32 labels the DNA since bacteriophages don't have phosphate -when the results were analyzed, after E.coli was infected, there was radioactivity due to the P32 labelled phages -the virus injects its DNA and the proteins stay on the outside -labelled nucleic acid enters the progeny -DNA acts as a template for replication, transcription, which create more viral proteins and other viruses -the proteins are all newly made 5) Fibre Diffraction Experiment -important for Watson and Crick to understand in order to make a model -Franklin determined a fibre diffraction pattern for DNA which showed a helical structure -there is a fibre diffraction pattern because the pattern of DNA is floppy and not rigid -models and crystal structures of small pieces of DNA (i.e.: tetramers, which can be crystallized) as well as the data from the fibre diffraction were used to determine structure of the standard model known as the B-DNA model Is DNA structure always the same? -the DNA structure is not a uniform helix; may be caused by ionic strength in the fibres -regions that are close together are known as the minor groove -there are molecules that can fit into the minor groove and interact with the bases -the regions that are farther apart are known as the major groove -the major groove is the part that interacts with proteins to regulate genes -proteins fit into the major groove -can form interactions with the base pairs (usually the pyrimidines) -width is about 2nm (20 angstroms) -the 2 strands are anti-parallel and read from 5' to 3' but in opposite directions for each strand -a copy of a DNA strand is not an exact copy -it involves using 1 strand as a template to make a complement through synthesis -the standard helix is "'right-handed" -as you look down the DNA structure, it turns clockwise (to the right) and away from the observer -the DNA structure is not always the same -you can get fibre diffraction patterns that are different (i.e.: under different ionic conditions) -e.g.: Z-DNA is associated with G-C rich DNA and is not right-handed -e.g.: A-DNA is thought to be the structure for dsRNA -is a right handed helix -e.g.: A-T rich DNA has narrower minor grooves -this suggests microhetrogeneity can occur within a long molecule within localized regions in DNA structures which can be associated with certain functions -caused by different ionic strengths in the fibres, amounts of A-T and G-C, salts, modifications in the DNA, and proteins that bind to DNA -we use B-DNA as the standard so that we can compare other structures to it Is the DNA structure in the cell relevant? -matrix consists of a group of proteins which bind DNA into loops (mostly associated with eukaryotic cells) -difference between RNA and DNA structure: -e.g.: tRNA has a primary sequence (5' GGGCGUG…etc.) -RNA is more compact and has regions that are hydrogen bonded to itself (5' bonded to 3') and this is known as a stem structure; there are loops which are regions with no hydrogen bonds -contains modified bases (represented by red dots and symbols) -secondary structure involves hydrogen bonding of the primary sequences (represented in flat space) -hard to predict from primary structure -tertiary structure involves the representation in space -hard to predict from primary structure -to form tertiary structure in tRNA, there is hydrogen bonding -in RNA, it has a hydroxyl which is used to make hydrogen bonding -quaternary structure involves RNA folding into proteins -if given a DNA sequence and asked for RNA sequence, this cannot be done unless it is assumed that there are no modified bases and no post-translational modifications -DNA shows similar structure along its along (as long as it is double stranded) -if it was single stranded it may look like an RNA but would not necessarily act like it because it lacks the 2'-hydroxyl -DNA can be extensively modified in the cell -modifications are post-replication -e.g.: the methylation of cytosine; 5MeC influences chromatin structure and gene expression (regulation of eukaryotic genes) -RNA can be extensively modified in the cell -modifications are post-transcription -e.g.: methylation of guanosine -e.g.: polyadenylation of mRNA -e.g.: RNA editing that changes cytosine to uridine -chemistry is important for structure and function -tertiary structure is important for the correct binding of proteins to create the quaternary structure -hard to predict tertiary structure in RNA -structures are dynamic Without chromatin, would it be necessary to invent it (chromatin)? -DNA organization in eukaryotes and prokaryotes: -in eukaryotes, the protein that binds DNA to form the first level are known as histones -histones are also involved in different levels of organization -are the basic, repeating structure for every unit -there are also non-histone proteins involved in gene regulation and expression; involved in forming a matrix / scaffold -in prokaryotes, DNA is organized more simply, but follows the same basic principles -the basic principles: -DNA packaging involves specific proteins and are achieved via repeating subunits -the same type of unit (histones) is used repeatedly -compacting DNA involves using histones to wrap them -transcribing DNA involves taking the DNA off the histones -more compact structures are based off simpler structures -subunit assembly must be dyn
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