Exam 1: Lecture 5 Flashcards Preview

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Chromosome Structure in Cell Cycle

-varies during cell cycle
-interphase: de-condensed so replication enzymes will have access to DNA strands.
-prophase: re-condense so they can be sorted into two daughter cells quickly
-cycle of condensing and de-condensing takes place during each round of mitosis



-genome of human cell when stretched is 2 meters long while the diameter of a nucleus is about 5 microns.



-mediates the packaging of DNA into tight discrete structures (metaphase chromosome)
-DNA tightly packaged around histones
-DNA is negatively charged due to phosphate groups so the charge of the histone proteins at any one time in the cell cycle is key to determining if chromosomes are being condensed or de-condensed.
-positively charged histone will interact more tightly with DNA than negatively charged one
-addition of phosphate, methyl and acetyl groups by modification enzymes can alter overall charge of histone proteins


Nucelosome Composition

-DNA + nucleosome octamer
-chromosome condensation during prophase involves the local winding of the DNA double helix around an octamer (DNA-histone core) of histone proteins


Methyl Modification of Histone Code

-neutral effect on overall charge of histones, however makes histones more hydrophobic.
-nucleus is water based solution so histones try to package more tightly.
-enzymes that add methyl groups onto histones are Histone Methyl Transferases (HMT)
-key protein domain found in all HMT's is the SET domain



-different ones differ in their substrate specificity and in the number of methly groups (1, 2, or 3) that are added to amino acid residues within the histone protein.


Methylation (Where?)

-occurs mainly at lysine and arginine residues


Removal of Methyl Groups

-mediated by demethylation or demethylimination
-in first, only methyl gorup is removed
-in second, methyl and adjoining amino group are removed


Phosphate Modification of Histone Code

-addition of negatively charged phosphate groups to histone core proteins makes overall charge more negative.
-leads not t ode-condensation of chromosomes, but to condensation


Most Common Phosphoylation

-additon of phosphate group to serine 10 at histone H3.
-initiates G2 of interphase, reaches a maximal at metaphase and then diminishes during anaphase and telophase.
-thought that phosphorylated H3 does cause small local loosening of DNA from histone core just enough to allow condensation protiens to interact with nucleosome and force chromosome to package even more tightly



-enzymes that add phosphate groups to histone core proteins
-Histone Phosphoryltransferases


Acetyl Modification to Histone Code

-addition of acetyl groups (which are negatively charged) makes the overall charge of the histone core more negative
-evidence to suggest that acetylation of histone H3 and H4 accompanies and may play a role in the de-condensation of chromosomes in late telophase and interphase
-removal of acetyl groups from histones is thought to promote DNA condensation in late G2 and prophase.



-enzymes that add acetyl groups to the histone core proteins
-Histone Acetyltransferases



-enzymoes that remove acetyl groups from teh histone core proteins
-Histone De-acetylases


Histone Acetylation, Methylation and Phosphorylation role in Transcription

-local loosening or tightening of the DNA double helix around the histone core will influence the accessibility of DNA binding proteins to promoter and enhancer sequences
-genes can be turned off if transcription factors are denied access to these sites. Conversely, if the grip that DNA has on the histone core is loosened then DNA binding proteins can find the promoter and initiate transcription.
-histones are naturally more positively charged so pulling off of acetyl/phosphases/methyl transform from gound state to something more highly or loosely wound.


Histone Modification: Intra nuclesomal Interactions

-some modifying enzymes can add multiple copies of the same group to a single histone
-In other cases a single enzyme can add two different groups to two different histones. Can occur if the modifying enzyme of interest has two different protein domains (ie a SET and a HAT domain)
-two different modifying enzymes can add different groups to different histone proteins. Special cases in that the modifying enzymes are part of a larger protein complex.
-all these examples describe situations in which the modifying enzymes are added groups to histone proteins within a single nucleosome


Higher Order Packaging of Chromosomes Requires Histone H1

-each nucleosome consists of the histone octamer and 146bp of DNA
-Between adjacent nucleosomes is approximately 20-60bp of naked linker DNA. This level of packaging is insufficient to generate the condensed chromosome that we see at metaphase so additional higher order packing is required.
-histone H1 protein binds to the linker region and brings each neighboring nucleosomes closer to each other regulated by phosphorylation and peak point is early mitosis
-several additional levels of chromosome packaging however these do not use histones. Instead these higher order events use condensation protein such as Topoisomerase II and condensins
-Histone H1 is not part of the histone core


Histone-DNA Interactions

-core histone proteins interact with DNA double helix at minor groove which is determined by charge and not sequence
-remember DNA double helix negatively charged while histones are naturally charged due to lysine and arginine residues.
-ability of histones to interact with DNA in a non sequence-specific manner allows for nucleosomes to form along the entire length of the chromosome
-transcription factors interact with DNA at the major groove and bind to specific sequences and this restriction prevents transcription factors from binding along the entire length of the chromosome.


HIstone-Histone Interactions

-the four histone core proteins (H2A, H2B, H3 and H4) all contain a Helix-turn-Helix-turn-Helix motif
-In this domain three alpha helices are separated by short stretches of amino acids and has two biochemical roles
-many transcription factors use the Heilx-turn-Helix motif to interact with the major goove of DNA.
-however, histones use the extended HTHTH motif to mediate histone-histone interactions
-The following histone protein interactions are mediated by these motifs (H2A-H2A, H2B-H2B, H2A-H2B, H3-H3, H4-H4, H3-H4, H4-H2B and H3-H2A)


Alpha Helicies

-naturally all binding proteins have alpha hellicies that bind into major groove
-alpha helicies used to hold proteins together


HIstone Remodeling

-ultimate goal is to loosen the charge interaction between histone core proteins and negative DNA
-accomplished through several experimentally proven models for how nucleosoems are remodeled


Necleosome Remodling (1st Model)

-DNA double helix slips around histone core thereby allowing access to certain sites along chromosome (thought to occur most often during transcription)


Nucleosome Remodeling (2nd Model)

-nucleosome completely removed from one segment of DNA and transferred to a second DNA strand
-thought to occur frequently during DNA replication as well as during transcription


Nucleosome Remodeling (3rd Model)

-localized DNA conformational change
-grants access to limited DNA sites without wholesale changes in nucleosome architecture.
-limited to transcription


Rubinstein-Taybi Syndrome

-problems with a human HAT protein
-geneotype: mutations in human Creb Binding Protein (CBP)
-mental retardation and seizures, absent/extra kidney, skeletal and muscle



-multi-domain protein that is involved in histone modifications during transcription.
-histone acetyl transferase (HAT).
-in absence of HAT activity, CBP is unable to acetylate histones so DNA does not loosen from histone
-decrease in transcriptional levels of many genes



-octamer includes dimers of the following 4 histone proteins: H2A, H2B, H3 and H4.
-each of these extend N-terminal tails outside of DNA loop.


Modification of Histone Proteins

-phosphate, methyl, and acetyl groups are added by modifying enzymes to N-terminal histone tails which changes the overall charge of the histone core proteins
-during prophase the histone core proteins are modified so that the overall charge is positive
-nucleosome formation is promoted since DNA is naturally negatively charged
-During S phase histone core proteins are modified so the overall charge is negative
-DNA repelled from histone core and chromosome de-condenses along entire length.


Histone Modification: Inter-nucleosomal interactions

-situations in which single proteins can add groups to histones on adjacent nucleosomes (adjacent briding) or on distantly located nucleosomes (discontinuous bridging)
-similar rules appply
-in some cases modifications being added are the same but some circumstances a single enzyme can add two different types of modifications