weber uiuc edu 24 September 2007 MCB150 Lecture 13 Lecture 13 24 September 2007 Announcements More broken hand notes Exam I rated o We were close to average for the history of this class o He hoped for better because we had more time on the questions o Wants to know why the scores weren t high enough o We should not lean to heavily on the homework problems for studying it may not give a strong enough base to the exam o He has no reason to believe that we are not doing the homework properly o Do not take the easy way out on the homework problems o Be reading the material thinking about the material Review the answers to the exam and understand why the answers were wrong This is a completely new unit The material from exam I will not be tested explicitly but the rest of the exams depend upon our knowledge of the first exam Make some study changes DNA Organization How is DNA distributed throughout the cell We know that nucleotides make up the nucleic acid That strand of DNA will come into proximity of a complementary piece of DNA and will form a double helix with hydrogen bonding Will form chromatin a mixture of DNA and protein A unique piece of chromatin is a chromosome o Some are circular prokaryotes typically mitochondria chloroplasts o Some are linear eukaryotes typically We have lots of linear DNA molecules Except in the mitochondria which are circular Organellar DNA can be circular Chloroplasts in plants have circular chromosomes too o Note the distinctions Packing of DNA How do we pack DNA into the cell or nucleus o In bacteria the DNA when unwound is 1000x the length of the cell itself o In humans it is even more extreme length over 2m in length Obviously this must be packed tightly yet remain accessible Space averages 1 10um in diameter Bacterial Chromosome Organization Bacterial chromosomes are supercoiled o Think of doing this with a rubber band coil it around itself to tighten it Catalyzed by topoisomerases o It is a reversible reaction o Can twist multiple times Topoisomerase makes a cut in the covalent linkage in the phosphodiester backbone Page 1 of 4 weber uiuc edu 24 September 2007 MCB150 Lecture 13 Spins the DNA around then ligates the cut bond o Uses a molecule of ATP to drive the reaction There are different families of the topoisomerases o Some wind in one direction others in another etc A relaxed bacterial molecule in the figure is long The supercoiled DNA takes up less 3D space o It can be coiled even more tightly than pictured o The coils are not covalently bonded just crossing over o Can simulate this with two pieces of string wind them around each other loop and tape Then cut one of them and twine it more tightly Eukaryotic Compaction and Compaction The picture is of a plant cell How do you get 2m of DNA into a 5 8um diameter nucleus Roger Kornberg in 1974 discovered how it was done o Cracked open the cell of a eukaryote o Extracted the nucleus o Removed the chromatin o Isolated DNA and protein associated with it chromatin o He noticed the beads on a string His idea is that the DNA is wrapped around beads The string is the DNA the beads are protein Chromatin Structure Isolated the DNA chromatin Digested the DNA with nucleases gentle ones o DNases enzymes that digest DNA o He chose DNase that would digest the string between the beads o The DNase could not get to the DNA around the beads o Thus linker DNA is digested The fundamental unit of DNA organization the nucleosome or the core nucleosome particle o Diameter is 10nm actually 11nm o He then dissociated the string from the bead using a salt concentration to determine the protein content and DNA base pairs o In the nucleosomes there are 146 base pairs 146bp wrapped roughly twice around eight proteins Note in WebCrossing o The text Cooper says 147 bp We will stick with 146 bp o If you consider chromatosomes which include the histones in the core see next slides the H1 etc you get 166 bp o If you add in the linker DNA then you get 200 bp o We need to know these numbers Chromatin Proteins Histones The proteins are called histones Relatively small 100 amino acids Very basic Rich in basic amino acids those with positive charges like lysine Why Page 2 of 4 weber uiuc edu 24 September 2007 MCB150 Lecture 13 o Because DNA has an overall negative charge due to the negative charges on the phosphates in the chain o It makes sense that you would wrap the DNA around the positive charges Names H1 H2A H2B H3 and H4 o These are found in almost all eukaryotes H4 is 102 amino acids in length The primary sequence in cow H4 and pea plant H4 there are only two amino acid differences the other 100 are the same o Therefore histone protein peptide amino acid sequences are very highly conserved This is an indication that changes would be severe Bacteria do not have histone proteins o Bacteria have histone like proteins Archae do have histones o This is more evidence that they are closer to eukaryotes o Similar in sequence to ours but sufficiently different Nucleosome The 8 proteins consist of 2 each of H2A H2B H3 and H4 146 base pairs are wrapped around these 8 Diameter is 10 nm 11nm These are called 10nm fibers Nucleosome Structure The positives do attract the negatives but it isn t sufficient to hold it all in place To hold it the remaining histone H1 is a linker histone o H1 is not a core nucleosome histone Instead it acts like a piece of tape holding it all together The color figure shows the 3D structure of a histone o The helices play a big role in the structure o You can see the histones easily on the left while on the right you can see the way the DNA is coiled about Note the end terminal tails of the histones o Not all histones have tails o These are regions of the primary sequence that don t have much appreciable secondary structure o They tend to stick out away from the core o The side chains of these amino acids are receptive to chemical modification See below o The chemical modifications will influence how the tails interact with each other and with the rest of the chromatin o We will see that these tails play a role in packing DNA Cells can modify these histone tails by covalent but reversible chemical group addition or removal of methyl groups acetyl groups or phosphate groups These changes look different in 3D space These combinations each say something different Modification will alter the level of packing Page 3 of 4 weber uiuc edu 24 September 2007 MCB150 Lecture 13 This is called the histone code The code is