Proteins and Chromatin Structure Flashcards
amino acid side chain (R group) properties
- shape, folding
- protein charge (local and overall)
- enzymatic properties
- modification sites
- H bonding properties
- hydrophilic vs hydrophobic
general amino acid structure
amine group (+ve)
alpha carbon (connected to R group)
carboxylic acid carbon (functional carbon)
proteins sorted by R group: categories:
- acidic
- basic
- nonpolar
- polar (uncharged): H bonds
- aromatic
which protein/gene always at start of protein seq?
methionine (Met)
AUG/ATG
protein primary structure
- bonded by peptide bonds (covalent), H2O = byproduct
protein secondary structure
H-donor, O-acceptor
held by H bonds only
protein tertiary structure
(need in order to be functional)
- hydrophobic parts tend to clump inside
quaternary structure
multi-subunit protein
- tertiary structures are its subunits
diff protein functions
- defense
- transport
- communication
- storage
- enzymes
- structure
protein activity control
first step: post-translational modification (covalent) and transport
- not dictated by DNA or genome (unlike primary->quaternary structures)
- epigenetic
- N-terminal like 5’ end of gene
(N-terminal TO C-terminal)
polypeptide means
unfolded proteins
primary (structure) means?
nascent
native = functional; nascent = unfunctional
protein modifications for transport
(modifications necessary for transport from cytoplasm, where translation takes place)
- proteins that are membrane bound or destined for secretion (e.g., receptors and protein hormones) are synthesized by ribosomes associated with ER membranes
– the ER associated with ribosomes is rough ER (RER)
- this class of proteins all contain a N-terminus signal seq or signal peptide; transport is co-translational (N-terminus starts entering ER WHILE rest of peptide still being made by ribosomes)
- proteins destined for organelles: diff signal seq at N-terminus
protein modifications (11)
- proteolytic cleavage
- phosphorylation
- sulfation
- acylation or acetylation
- glycosylation
- methylation
- prenylation
- vitamin C-dependent modifications
- vitamin K-dependent modifications
- ubiquitin (ubiquitination)
- control of protein stability
proteolytic cleavage
proteolytic cleavage: most proteins undergo this following translation
- simplest form - removal of first methionine
phosphorylation
phosphorylation
- post-translational phosphorylation one of most common modifications
- occurs as a mechanism to regulate protein activity
– transient (non permanent): phosphate (1 or more) is added and later removed (or transferred to other protein); gives 2 -ve charges per phosphate
- enzymes involved:
– kinases: transfer a phosphate group form donor (ATP) to acceptor (protein) (adds phosphate)
– phosphorylases: transfer a phosphate group form an inorganic phosphate (adds phosphate)
– phosphatases: remove phosphates
in animal cells, SERINE, THREONINE, and TYROSINE are AAs subject to phosphorylation (-OH group = phosphorylation site)
sulfation
- sulfate modification of proteins occurs at tyrosine residues
- since sulfate is necessary for biological activity, it is added permanently and NOT for regulatory modificaiton
acylation or acetylation
- in most cases, acetyl group is added to N-terminal AA (after first Met is removed)
- Acetyl-Coa is acetyl donor
Glycosylation
- glycoproteins consist of proteins with covalently linked sugars
– consensus AA for addition: Asn - X - Ser/Thr
(X is any AA except Pro)
Asn = asparagine
– sugars are modified
– major mechanisms for cell surface identification
Methylation
- post-translational methylation occurs at lysine residues in some proteins
- activated methyl donor is S-adenosylmethionine
- ex. cytochrome c and calmodulin
Prenylation
- addition of compounds derived from cholesterol biosynthetic pathway, hydrophobic fatty acid chains (membrane anchoring)
- can make more non-polar
- ex. Ras
Vitamin C-dependent modifications
- modifications of proteins that depend on vit C as a cofactor
- ex. collagens and peptide hormones like oxytocin and vasopressin
Vitamin K-dependent modifications
- vit K is a cofactor in carboxylation of glutamine residues
– carboxylation adds a carboxyl group - protein can chelate calcium ions (chelate - arresting)
- ex. some anticoagulants
control of protein stability
- some proteins are rapidly degraded, whereas others are highly stable
- specific AA seq in some proteins have been shown to promote rapid degradation (recognized by peptidases/proteases)
ubiquitin (ubiquitination)
- covalent post-translational addition
- addition of ubiquitin protein via Lys residues (7 Lys)
- Mono-: can be a location signal for membrane transport
- Poly-: signal for proteolysis
DNA, RNA, protein structure summary
- nucleic acids use nitrogenous bases (DNA -> RNA = transcription)
- proteins use amino acids (mRNA -> protein = translation)
- DNA is highly regulated, rarely modified, relatively permanent - very few intentional modifications after replication
- RNA is highly regulated, modifiable, transient
- protein is highly regulated, modifiable, transient
chromatin = ?
protein + DNA
chromatin structure (E. coli example)
diameter of bacterial cell is around 1nm; DNA is around 4 mil bp (1.36 mm long DNA thread which is 2nm wide)
bacterial “chromosome”
- genome forms a compact structure = nucleoid (mixture of supercoiled and relaxed regions)
- DNA organized in 50-100 loops (domains)
- circular molecule is compacted by association with:
– polyamines
– HU (heat unstable) proteins
– supercoiling
polyamines
(spermine and spermidine, +ve charge) - NOT proteins, molecules
HU (heat unstable) proteins
(small, basic, +ve, dimeric) and H-NS (histone-like nucleoid structuring) (monomeric, neutral) DNA binding proteins
supercoiling
can occur in space or around proteins
- unrestrained - path is supercoiled in space and creates tension
- restrained - path is supercoiled around protein but creates no tension
avg of euk chromosome?
150 Mbp = 150 million bp
there are ___ chromosomes in human somatic cell
46
approx ____m of DNA/human cell
2.5
avg size of eukaryotic cell =
10-100 micrometers
eukaryotic chromatin (chromosome, nucleoprotein, pulsed-field gel electrophoresis. dark field electron microscopy)
- eukaryotic genomes organized to form linear chromosomes
- each chromosome has single, linear DNA molecule
- nucleoprotein material of eukaryotic chromosome is chromatin
- individual chromosomes can be separated by pulsed-field gel electrophoresis (one band = one chromosome)
– electric field repeatedly alternated
– AND by dark field electron microscopy, fiber structure of a chromosome resembles “beads on a string”
chromatin organization
“beads on a string”
- fundamental unit of organization of chromatin = nucleosome
- each nucleosome has a CORE PARTICLE of histone proteins, that are wrapped by DNA
- also: non-histone proteins = important for chromatin organization
what is nucleosome?
nucleosome core + core DNA
chromatin organization: histones
- in all eukaryotic nuclei
- small proteins rich in lysine and arginine (at normal pH their “extra” amino groups become NH3+) - basic, +ve proteins
– electrostatic interactions
– N terminals wrap around DNA
histone’s 5 major subunits
- H1 (linker), H2A, H2B, H3, H4
– H2A, H2B, H3, H4 form a octet complex (8 proteins)
H1 (linker) is in nucleosome core (T/F)
False
how does histone octet in nucleosome core form? what does linker (H1) do?
- H3-H4 tetramer
- H2A-H2B dimer (NOT tetramer on its own, need H3-H4 tetramer)
- linker histone -> on side, packs DNA closer to nucleosome core
chromatin organization: nucleosome
- nucleosome = octet of histones and wrapped DNA
- DNA (147 bp) wraps around octet approx 1.65x
- H1 associates with DNA and octet in linker region -> binds two distinct regions of DNA duplex
nucleosomes are uniformly distributed (experiment)
- low conc micrococcal nuclease (don’t want full digestion)
- only linkers (DNA) were cleaved (nucleosome is too packed to cleave)
- realized nucleosomes are uniformly distributed
chromatin organization (fiber length)
- 10-11 nm fiber
– likely a consequence of unfolding during extraction in vitro
– H1 NOT required - 30 nm fiber
– basic constituent of interphase chromatin, mitotic chromosomes
– H1 required
chromatin organization: fibers
- core histones alone: ~4-fold compression
- core histones + H1: 25- to 100-fold compression
- nucleosome is 10-11 nm in diameter
- 10 nm fiber re-packed into a 30 nm diameter fiber
- higher salt conc during isolation (stabilizes DNA) - diff forms (10 nm nucleosomes - beads
-> more condensed 30 nm fibers) - recent electron microscopic studies - dynamic structure
chromosome organization: morphology
- 30 nm chromatin fiber condenses to metaphase chromosome = 1400 nm
- attachment of chromatin fibers to non-histone protein complexes = scaffold (genes in scaffold loops)
- nucleosome fibers condense more into 30 nm chromatin fiber. different theories about its form (solenoid or zig-zig). pre-dominant form in interphase nucleus
- duplex DNA winds around histone octamers to form nucleosomes = 10-11 nm histone fiber
- primary structure of DNA = duplex helix = 2 nm duplex
chromatin is organized in distinct CTs, meaning?
chromosome territories
chromatin has ___ and ___
loops and domains
chromosome structure (euchromatin/heterochromatin)
euchromatin
- less tightly packed, consists of transcriptionally active DNA, susceptible to DNase digestion
heterochromatin
- tightly packed, less susceptible to DNase digestion and transcriptionally inactive
– constitutive heterochromatin - highly condensed inactive chromatin; consists of repetitive DNA, very few genes (constitutive = always)
— e.g., centromere (specific seq, attachment point for sister chromatids and spindle fibers
—- e.g., telomere (end of chromosome)
– facultative heterochromatin - not active in any particular tissue. forms under specific circumstances and/or certain tissues to silence gene expression (facultative = optional; sometimes euchromatin becomes heterochromatin)
— X-chromosome inactivation (Barr body formation)
— imprinting
chromatin elements and centromeres
chromatin elements (elements = particular nucleotide seq, DNA regions)
- locus control regions - shared control regions (usu upstream form gene clusters) - control chromatin condensation
- matrix and scaffold associated regions - mostly AT-rich DNA which anchors to nuclear matrix (AT easier to unwind than GC)
- insulators - regulatory domains in DNA - define domains of gene expression (can block enhancers from approaching gene)
centromere = chromosome region which contains site of attachment for spindle fibers
- kinetochore = centromere + protein (connect to fibers)
- in situ hybridization of metaphase chromosomes shows satellite DNA at centromeres (highly repetitive seq)
telomeres
- specialized DNA regions at chromosome ends
- repetitive seq
- protect chromosomes from shortening during replication and from degradation (by “looping” of 3’ overhang)
chromatin organization: non-histone proteins
- matrix attachment regions (MARs) or scaffold attachment regions (SARs)
- DNA elements bound by scaffolding riboproteinaceous structures
– suggested: this binding is required for replication and transcription
– MARs A:T rich but no universal (consensus) seq
– may incl cis-acting sites that regulate transcription
– usu recognition site for topo II (role in packing) - SMC proteins (responsible for scaffolding)
- DNA replication proteins
- transcriptional factors, chaperone proteins, etc
chromatin organization: histone proteins
- highly conserved proteins esp H4 (important for living)
- H1 least conserved, most variations
- H5 = extreme variant of H1
- specialized: H3 variant specific for centromeres - CenH3
nucleosome disassembly and reformation
- replication of DNA requires particle disassembly of nucleosome
- newly replicated DNA immediately packed: first bind H3-H4 tetramer and THEN two H2A-H2B dimers, H1 is last
- H1 -> tighter DNA wrapping
- more DNA (from replication) - newly synthesized histones are needed
- old and new histones present on both daughter chromosomes
- chaperone proteins - -ve charged proteins, assist assembly of histones (escort dimers to replicating target DNA); negative bc they repel DNA and bind to histones
- replicating DNA is ABSOLUTELY NECESSARY for nucleosome assembly
chromatin organization: histone tails
- histones have N-terminal tails extending out from nucleosome - several +ve lysines (basic AAs)
- protruding tails integrate into “grooves of screw” - directly wrapping around DNA
- tails are required for formation and stabilization of 30 nm fiber through interaction with adjacent nucleosomes
transcriptionally active genes and chromatin condensation
- eukaryotic transcription and replication occur in context of chromatin:
chromatin modification necessary for replication to start and change gene expression: - transient modification of AAs in HISTONE TAILS
modifications of histone tails
- acetylation of histone tail (lysine) generally associated with active gene expression (repels DNA when added, leads to local unwinding)
- ubiquitinylation of histone tails (lysine) - mono = non-destructive modifications (same as ubiquitination)
- methylation of histone tail (arginine and lysine); may be associated with active or inactive genes
- phosphorylation (Serine) - generally assocaited with active gene (not very clear b/c histones are phosphorylated during mitosis)
- methylation and acetylation of lysine and phosphorylation of serine REDUCES overall +ve charge of protein
histone code hypothesis
- serial modifications of histones’ tails are “landmarks” for proteins which read chromatin (domains recognize modified tails)
- creates “open” chromatin necessary for transcription, replication, repair, and recombination
___ binds to methylated lysine of histones
chromodomain
____ binds to acetylated lysine of histones
bromodomain
chromodomain and bromodomain are not proteins. they are ____
parts of proteins
what do methylation and acetylation of lysine AND phosphorylation of serine do?
reduce overall +ve charge of protein
-> keep DNA loose (from -ve repel)
acetylation and deacetylation of histones’ tails (details, enzymes)
- enzymes: histone acetyltransferase (HAT) - catalyzes forward equation, promote gene exp)
- histone deacetylase (HDAC) - catalyzes reverse equation, suppress gene exp)
Histone + Acetyl-Coa <-> Acetyl-Histone + coenzyme A
- acetylated form is negative (H+ atom replaced by -acetyl group)
– affects chromatin’s condensation
– necessary for activation of transcription - nucleosome free regions - contain actively transcribed DNA (gene exp in progress) - DNase-sensitive regions
DNase I test
- isolate nuclei, treat with diff conc DNase
- separate protein, DNA
- digestion, gel electrophoresis, southern blots
2 possibilities:
1. no signal - gene expression was in progress; DNA was free of histones, available for digestion by DNase
2. signal - no gene expression (too tight, couldn’t cleave)
transcription assay
- usually involved “naked” nucleosome-free DNA, since usu in vitro
(naked DNA = DNA w/o proteins
prok vs euk
prok:
- no membrane-bound nucleus
- single, circular chromosome
- no membrane-bound organelles
- single cell organisms
euk:
- membrane-bound nucleus
- linear chromosomes (23 pairs in humans)
- have membrane-bound organelles
- single or multicellular
extrachromosomal means
not in chromosome
ex. plasmid, mitochondrial DNA
human chromosomes:
7 groups of autosome (classified by size), 2 groups of sex chromosomes
