Block 1 Flashcards
The Chemical Composition of Nucleic Acids
Basic Components
o The nitrogenous bases
♣ Purines: Adenine and Guanine (each has two aromatic rings)
♣ Pyrimidines: Cytosine, Thymine, Uracil (each has one aromatic ring)
o Ribose or Deoxyribose (the 2’ C is deoxygenated in DNA, has -OH in RNA)
o Phosphate groups
Polynucleotides
o Individual nucleotides are joined to each other at the 3’ and 5’ carbons through a phosphodiester bridge to form the sugar-phosphate backbone of DNA
o Nucleic acid chains have polarity
♣ One end of a chain has a 5’ PO4 group
♣ One end has a 3’ –OH group
♣ By convention, base sequences are written in the 5’ 3’ direction
Right handed, B-form DNA
the most common conformational form of DNA
Major and Minor Grooves:
In the most common form of the double helix (B-DNA), the glycosidic bonds of a base pair are not diametrically opposed to each other, resulting in major (12 angstrom wide) and minor (6 anstrom wide) grooves
♣ The grooves are lined with potential hydrogen bond donor and acceptor atoms
o Many DNA binding proteins bind in the major groove of DNA in a sequence specific manner
Clinical application of DNA structure (grooves)
♣ CLINICAL APPLICATIONS: Certain cancer drugs (actinomycin D) can exert their effects by intercalating into the minor groove to interfere with with RNA and DNA synthesis
• The antibiotic actinomycin D (produced by Streptomyces antibioticus) binds to DNA duplexes, thereby interfering with the action of enzymes engaged in replication and transcription
• Actinomycin D is an anti-cancer drug commonly used in the treatment of pediatric malignancies such as Wilms’ tumour, Ewing’s sarcoma, and rhabdomyosarcoma
Key features of DNA structure
o Two helical polynucleotide chains coiled around a common axis that run in antiparallel directions (resembles a twisted ladder structure)
o Bases are on the inside of the helix and phosphate sugar backbone is on outside
♣ The planes of the bases are “stacked” perpendicular to the helix axis
♣ Stacking resonance (sharing e-) helps stabilize the double helix
o Helical structure repeats after 10.5 residues at intervals of 36 anstroms
o Two chains of helix are held together by hydrogen base pairing
♣ Adenine must pair with thymine (A-T: two bonds) and guanine must pair with cytosine (G-C: three H bonds)
• A-T bonds are weaker than G-C bonds… 2 < 3
3 Forces Holding DNA Together
3 Forces holding DNA together:
- Resonance
- H-Bonding
- Ionic Bonding
Non-B Form DNA
o The classic Watson-Crick B-DNA is an average structure
o In vivo, DNA has subtle but functionally significant deviations from this average structure
Z-DNA
♣ Z-DNA (left-handed, zigzagging alternating purines and pyrimidines, may have a role in gene expression)
• Z-DNA binding proteins required for pathogenesis have been isolated from poxviruses, including variola (agent of smallpox)
♣ Additional conformations: triplexes, cruciforms, slipped structures, etc.
Properties of DNA/RNA: Denaturaton/Renaturation
o The two strands of a double helix separate when hydrogen bonds are disrupted by changes in pH or heating
♣ Ionic composition (salt concentration) of the solution will effect denaturation and annealing rates
♣ Size can play a role, mostly in extremes (super long vs super short)
o Denaturation (melting) and renaturation (annealing) can be monitored spectrophotometrically
♣ DNA melting can be monitored spectrophotometrically at 260 nm dur to a hyperchromic shift that occurs upon base stacking annealed DNA
• Bases absorb UV light better when ss VS ds
Melting Temperature
♣ The melting temperature (Tm) is defined as the temperature at which half (50%) of the helical structure is lost
• Tm depends on % GC content: DNA high in GC content melts at a higher temperature than NA with high AT content
• Tm depends on ionic strength of the solution: high salt favors duplex, low salt favors denatured
o Electrostatic repulsion caused by the charge on the phosphate backbone
Factors Affecting Tm
Inc GC = Inc Tm
Inc salt = duplex
Dec salt = denatured
B-DNA helix is flexible
o It can be locally bent, kinked, or supercoiled
o This flexibility is important for DNA compaction
DNA molecules can be: (conformations)
(1) linear (2) relaxed circular (3) supercoiled circular
o Linear and circular DNA have very different topological properties
o Also, there can be negative and positive supercoils based on direction of coiling
♣ DNA tends to be negatively coiled
Relaxed DNA has _ bp per turn of the helix
Relaxed DNA has 10.5 bp per turn of the helix
Positive supercoiling of DNA
o Positive supercoiling of DNA occurs when the right-handed, double-helical conformation (twisted in a right-handed fashion) until the helix begins to distort and “knot”
Negative supercoiling
o Negative supercoiling involves twisting against the helical conformation (twisting in a left-handed fashion), which preferentially underwinds and “straightens” the helix at low twisting stress, and knots the DNA into negative supercoils at high twisting stress
♣ Negative supercoils favor local unwinding of the DNA, allowing processes such as transcription, replication, and recombination
• DNA in cells is typically somewhat negatively supercoiled
Topoisomerases
enzymes that change the topological state of circular DNA but not its covalent state
Type I topoisomerases
o Type I topoisomerases create transient single-stranded breaks in DNA
♣ They are nicking-closing enzymes and can relax both negatively and positively supercoiled DNA
♣ The enzyme acts by:
• STEP 1: Cleaving one strand of DNA
o Active-site Tyr attacks a phosphodiester bond in one DNA strand, cleaving it and creating a covalent 5’-phosphotyrosyl protein-DNA linkage
• STEP 2: Passing a segment of DNA through the break
o Enzyme changes to an open confirmation and the unbroken DNA strand passes through the break in the first strand
• STEP 3: Resealing the break
o Enzyme in closed conformation; liberated 3’-OH attacks the 5’-phos-photyrosyl protein-DNA linkage to religate the cleaved DNA strand• DO NOT need energy donors (ie, ATP-independent)
• At each step, one high-energy bond replaces another
Type II topoisomerases
o Type II topoisomerases create transient double-stranded breaks in DNA
♣ The enzyme acts by:
• STEP 1: Two strands are cleaved
o The multisubunit enzyme binds a segment of DNA molecule, and a second segment of the same DNA molecule is bound at the N gate
o The second segment of DNA is trapped, and the segment is cleaved on both strands to form two 5’-phosphotyrosyl linkages to the enzyme
• STEP 2: The DNA is passed through the break
• STEP 3: The break is resealed
o The broken DNA is religated, and the second DNA segment is released through the C gate
• DOES require two ATPs to complete a reaction cycle
CLINICAL IMPLICATIONS of Superhelicity
• Topoisomerases are targets for antibiotics
o Coumarins (novobiocin, coumermycin A1)
♣ Inhibit bacterial type II topoisomerases and DNA gyrase from binding ATP; not often used to treat infections in humans
o Quinolones (nalidixic acid; ciproflaoxacin, Cipro)
♣ Inhibit the last step of topo reaction, which is resealing the DNA strand breaks
♣ Wide-spectrum and mostly selective for bacterial enzymes
• Topoisomerase inhibitors used as chemotherapy agents
o Targets cancer because most rapidly growing cells (tumors, others) express topoisomerases
♣ Rapidly replicating cells such as cancer cells have elevated levels of Type II topoisomerases, and are therefore more likely to incur lethal DNA damage through inhibition of Type II topoisomerases than slow growing cells
o Eukaryotic Type I topoisomerase inhibitors
♣ Captothecin, irinotecan (Campto), topotecan (Hycamtin)
♣ Trap the enzyme-DNA complex in its cleaved state
o Eukaryotic Type II topoisomerases inhibitors
♣ Doxorubicin (Adriamycin), etoposide (Etopophos), ellecticine
♣ Can block the binding of ATP
Chromatin
The genomic DNA within the nucleus in complex with its nuclear proteins – the mode by which the genes are complexed in chromatin determines which genes are activated and which are repressed
Heterochromatin:
highly condensensed, usually (not always) transcriptionally inactive (about 10% of the chromatin)
Euchromatin:
the remaining, less condensed chromatin; some, but not all, is transcriptionally active
The Nucleosome:
- The Nucleosome: the fundamental and smallest unit of chromatin packaging
a. There are millions of nucleosomes in each nucleus, each shaped like a rugby ball
i. Using electron microscopy, these can be seen as beads along a string (string being linear DNA)
ii. As in bulk chromosome, each nucleosome is half protein, half DNA
iii. The protein component of each beat consists of an octomer (8) of histone molecules, plus a few non-histone molecules
The Histones
i. Grouped within 5 related protein “families”
a. H1 family: termed “linker” histones because they are located between the nucleosomal beads
b. H2A, H2B, H3, H4: termed “core” histones
i. Two molecules of each core histone make-up each nucleosome; hence each nucleosome contains an octamer (8) of histone molecules
ii. All the histones are highly basic due to the enrichment of lysine+ and arginine+ residues, and are evolutionarily highly conserved, indicating their vital cellular function
Histone Function:
histones function to inhibit transcription, globally repressing the genome
Histone Acetylation
i. In order for transcription to occur, the histones must be modified via acetylation of the lysine+ and arginine+ residues to neutralize the positive charges
1. This results in repulsion of histones from DNA gene sites causing “chromatin opening,” a process that allows entry of transcriptional machinery
Histone Methylation
i. In contrast, methylation of histone H3 causes formation of the non-transcribable ‘hetero’-chromatin by serving as a binding surface for the non-histone protein HP1 (heterochromatin protein 1)
1. HP1 is bound, other proteins (i.e., transcription factors) can’t
Histone Phosphorylation
i. Phosphorylation of serine and threonine is usually associated with the repression of transcription that occurs during condensation of chromatin into recognizable chromosomes as the cell enters M-phase (mitosis) of the cell cycle
Posttranslational modifications of histones
Methylation and phosphorylation INHIBIT transcription
Acetylation ALLOW transcription
The posttranslational modifications of histones occur primarily at….
a. The posttranslational modifications of histones occur primarily at specific residues located within the histone amino-terminal tails protruding from the surface of the nucleosome
i. Histone modifications impact chromosome function though at least two distinct mechanisms:
1. Histone modifications alter the electrostatic charge of the histone resulting in a structural change in histones or their binding to DNA
a. This is exemplified by histone acetylation/deacetylation
2. Histone modifications provide binding sites for regulatory proteins containing protein domains that recognize specific histone modifications
a. HP1 uses its chromodomain to bind methylated lysines residues on histone H3 during heterochromatin formation
ii. Histone Modifications are carried out by specific histone modifying enzymes (histone acetyltransferases, methyltransferases, etc)
epigenetic modifications
i. Epigenetics modifications produce heritable changes in gene function that occur without a change in the sequence of DNA
DNA Methylation
- Another type of epigenetics modification is methylation of DNA, which is when a methyl group is added to the 5’ position of the pyrimidine ring in cytosine residues in CpG dinucleotides
a. DNA methylation is carried out by DNA methyltransferases (DNMTs)
b. This represses gene expression by blocking binding of TFs that normally activate genes
c. Can synergize with repressive histone modifications to promote heterochromatin formation
i. Methylated CpG in DNA is bound by methyl CpG binding proteins (MBPs)
ii. MBPs recruit histone deacetylases (HDACs)
iii. MBPs recruit histone methyltransferases (HMTs) - Methylated histones are recognized and bound by HP1, completely silencing gene
Clinical Relevance of DNA Methylation
a. CLINICAL RELEVANCE: Importance of epigenetic changes in diagnosis and treatment of cancer
i. Epigenetic modifications of DNA and histones can combine to suppress expression of tumor suppressing genes, whose products control cell growth by regulating the processes of mitosis and cell division
ii. DNMT inhibitors and HDAC inhibitors have been tested for use as anti-cancer agents
1. HDAC inhibitors directly inactivate HDAC enzymatic activity
2. DNMT inhibitors incorporate into DNA in the place of Cytosine, rendering CpG dinucleotides incapable of methylation by DNMT
Nucleosome Structure
a. Nucleosomes are approx. 50% (histone) protein, 50% DNA
i. Each nucleosome is considered to contain approx. 200 pb DNA
ii. Each nucleosome forms by combining two histone dimers (H2A/H2B) with one histone tetramer (2x H3/H4) to form the histone octamer
iii. Non-Histone: any chromatin protein that is not a histone (DNA repair enzymes, DNA and RNA polymerases, and TFs)
b. Transcribing chromatin does have nucleosomes!
i. Modifications during gene activation
1. The dissolution of one or two nucleosomes from the gene promoter and of histone H1 from linker DNA
2. Histone acetylation by histone acetyl transferases (HATs)
The 30nm Fiber
- The 30-Nanometer Fiber:
a. Six 200 bp nucleosomes are packed into a solenoid arrangement as one 1200 pb fiber with the help of Histone H1
i. Solenoid: 6 nucleosomes per turn
Loops
- Loops:
a. An average of 50 1,200 bp 30 nm fibers are coiled into a loop containing 60,000 pb of DNA
b. The bases of each loop are attached to a proteinaceous structure termed the nuclear matrix, which contains important structural and gene regulatory machinery
c. Looping circularizes the DNA, enabling localized genes to become supercoiled, which is required for transcription
d. Genes located in one loop can interact with up- and downstream regulatory sequences in adjacent loops, such as enhancers
Minibands
- Minibands:
a. Tandems of loops that encircle the chromosome axis
b. Proximity of loops on adjacent minibands would permit regulatory sequences in distal loops to interact
Mitotic Chromosome Structure
a. The formation of individual chromosomes at the onset of mitosis reflects hyper-condensation of chromatin, which is mediated by hyper-phosphorylation of chromatin proteins, especially histone H1
i. Each chromosome contains a single linear DNA molecule of an average 75 million bp DNA
ii. Centromere: locus of repetitive DNA in the middle of the chromosome
iii. Two telomeres: loci of repetitive DNA that cap the ends of the chromosome
Telomeres
i. Two telomeres: loci of repetitive DNA that cap the ends of the chromosome
1. Contain a repeating sequence of GGGTTA up to 15,000 bps
2. At each cell division, several repeats are lost in somatic cells causing shortening of telomeres and cellular senescence
3. Telomeres in germ-line cells are maintained at full-length via an enzyme termed telomerase, which adds on GGTTA segments after cell division
Clinical Significance of Telemeres
CLINICAL SIGNIFICANCE: telomerase is normally quiescent in somatic cells, but can be mutated and activated, causing abnormal maintenance of telomere length and cellular immortality that result in tumor formation
Nuclear Matrix
The nuclear matrix maintains the dynamic structural organization of the interphase nucleus by organizing the genome into domains that regulate gene expression and cell replication
Three Components of nuclear Matrix:
- Nuclear Envelope/Pore Complex/Nuclear Lamina
- Nucleolus
- Internal Nuclear Matrix
Three components of the Nuclear Matrix:
- Nuclear envelope/Pore Complex/Nuclear Lamina
Nuclear envelope
a. The nuclear envelope
i. A double membrane that encloses the perinuclear space, which is continuous with the lumen of the endoplasmic reticulum (ER)
ii. It is divided into segments, limited by nuclear pores
iii. The outer membrane (faces cytoplasm) contains ribosomes for protein translation
iv. The inner nuclear membrane contains integral proteins that bind the nuclear lamina, which in turn attaches to marginal heterochromatin
Nuclear Pore Complex
i. Nuclear pores between segments of the envelope are ~10 nm diameter
ii. The pore complex regulates passage of proteins into, and proteins/RNAs out of, the nucleus
1. Proteins smaller than ~40kD diffuse thru, larger proteins are under complex regulation
Nuclear Pore Structure
i. Each pore has three strata of proteins arranged as an octamer: (1) cytoplasmic stratum (2) nuclear stratum and (3) middle stratum
1. Strata contain ~100 different proteins termed nucleoporins
2. Nucleoporins in the cytoplasmic stratum serve as “docking sites” for proteins called nuclear cargo proteins that contain nuclear localization signals (NLSs)
a. NLSs are short amino acid sequences that contain positive charged basic amino acids (lysine [K] and arginine [R])
i. Cytoplasm Nucleus Import (only proteins are imported)
- Proteins larger than 45 kD must have a NLS to get in
- NLS-Receptor Proteins are in the cytoplasm
a. Example: importin, a heterodimer consisting of subunits importin- and importin-
i. Importin- binds the NLS of a nuclear cargo protein
ii. Then the complex migrates to the pore
iii. Importin- binds a nucleoporin in the pore complex
iv. Then, energy-dependent transport thru the pore
v. Once in the nucleus, the nuclear cargo protein is released from importin, which is cycled back thru the nuclear pore complex
Other mechanisms of nuclear import:
a. Proteins may have to be de-phosphorylated
b. Proteins may have to be released from a cytoplasmic masking protein to be recognized by importin
a.
ii. Nucleus Export Cytoplasm (Proteins and all RNA species are exported)
- Exportins mediate nuclear protein export
a. Exportins contain leucine-rich NESs (Nuclear Export Sequences) in their amino acid sequence
b. Many if not must nuclear proteins have both an NLS and an NES - RNA export does not depend on type of sequence of RNA
a. RNA export is mediated by RNA-BPs which may be bound to an exportin protein
i. Protein and RNA transport are not pore-specific
- The same pore can accommodate protein import as well as export of protein and RNA
Energy for nuclear import and export
i. Energy for nuclear import and export is provided by a GTP-BP termed Ran-GTP (“ran” in and out of the nucleus)
1. Within nucleus, Ran-GTP binds to both empty nuclear import receptors and cargo-bound export receptors, which migrate to the cytoplasm where Ran-GTP is hydrolyzed to Ran-GDP, releasing all bound moieties
a. Empty nuclear import receptors migrate back into nucleus
b. Nuclear import receptors bind proteins with NLSs before migrating back to nucleus
Clinical Significance of Nuclear Import/Export
i. CLINICAL SIGNIFICANCE: Drugs cannot readily penetrate the nucleus so nuclear structures are poor targets for therapeutic intervention
1. Knowing how substances are targeted to the nucleus, approaches may be designed that enable the development of drugs that can target specific nuclear structures and genes
a. The Nuclear Lamina
i. The nuclear lamina is subjacent to the inner nuclear envelope, to which it is bound
1. In interphase nuclei, the nuclear lamina maintains the nucleus as a sphere
2. During mitosis, the lamina breaks down due to hyperphosphorylation of lamina proteins
The nuclear lamina’s proteins
i. The nuclear lamina’s proteins are termed lamins A, B, and C
ii. Lamin A (LMNA) gene: Expresses Lamins A and C, which interact with the marginal heterochromatin
iii. Lamin B binds to the lamin-B receptor (LBR) on the inner nuclear membrane
1. When the nuclear envelope falls apart during mitosis, lamin B remains bound to the LBR, mediating re-formation of the nuclear envelope segments at the start of the next interphase
Clinical Significance of Nuclear lamins
i. CLINICAL SIGNIFICANCE: Laminopathies – mutations of the LMNA gene disrupt the lamina
1. Diseases range from several rare cardiac and skeletal muscular dystrophies to a severe form of premature aging (progeria), whch is caused by deletion of the part of the gene that generates the mRNA encoding lamina A
Nucleolus
a. The site of ribosomal RNA (rRNA) production, comprised of portions of ten chromosomes (each contributing ~40 rRNA genes)
i. Nucleoli do not have a limiting membrane
b. Cells need lots of ribosomes to live
i. To satisfy the cell’s demand, transcription of rRNA genes occurs constantly via RNA Polymerase I
1. Physical gene assumes the shape of a “Christmas tree”
The Internal Nuclear Matrix
- The Internal Nuclear Matrix (98% of the nuclear matrix)
a. Consists mostly of chromatin: DNA, Histones (H1, H2A, H2B, H3, H4) and non-histones (TFs, RNA and DNA polymerases, etc.)
b. Matrix proteins are disease-specific and tissue-specific
i. Some of the nuclear matrix proteins in normal cells and cancer cells is different – of diagnostic value (used in diagnostic tests for bladder cancer and cervical carcinoma)
a. Chromosomes occupying specific locations in the interphase nucleus
a. Chromosomes occupying specific locations in the interphase nucleus
i. Telomeres stick to the nuclear envelope while centromeres stuck to the opposite side of the envelope
ii. Chromosomes occupy the same site of the interphase nucleus, generation after generation
iii. Chromosome specific territories in the nucleus have been shown using fluorescent in situ hybridization (FISH) of DNA – also called chromosome painting
DNA replication and transcription occur in the …
a. DNA replication and transcription occur in the nuclear matrix
i. Although most nuclear material is removed during preparation of the nuclear matrix, sites of transcription are retained as indicated by in situ hybridization
a. The Nuclear matrix organizes DNA replication
i. In S-phase, DNA is replicated in groups of short sequences termed “replicons” – after matrix preparation, these replicons are retained in the matrix
Phases of the Cell-Cycle:
• Duration of each cell-cycle revolution is termed the generation time (Tg)
o During each Tg the cell goes through four cell-cycle phases:
♣ (1) G1 Phase (Gap 1) – duration is highly variable
• Cells in G1 have four choices:
o Senescence (i.e., G0)
o Differentiation (also G0)
o Apoptosis cell death
o Proliferation entry into cell cycle
♣ (2) S-Phase (Synthesis [of DNA] Phase)
• Chromosomes are duplicated, 2N 4N DNA
• Histones are synthesized to make-up the new chromosome
♣ (3) G2 Phase (Gap 2)
• Preparation for mitosis occurs; late in G2 the centrosome is duplicated
• Hyper-phosphorylation of histone and non-histone proteins occurs in late G2
♣ (4) M Phase (Mitosis):
• Cells become spherical
• Nuclear membrane disintegrates
• Chromatin condenses into chromatids (2/chromosome)
• Chromosomes align on equatorial plan, then chromatids segregate into “daughter” cells
Cell-Cycle Checkpoints
External Factors
• External Factors “1o messengers”
o Secreted molecules and peptides – GFs, cytokines, hormones – which interact with a cognate receptor that is in the cell membrane (or, for steroid/retinoid hormones, in the cytoplasm)
♣ Factors that ACTIVATE the cell-cycle (examples): FGFs, IGFs, Wnts
♣ Factors that INHIBIT the cell-cycle: TGF
Cell Cycle Checkpoints Internal Factors
• Internal Factors “2o messengers”
o “Early Response” genes: c-myc, fos, and jun
♣ Rapidly (15 min) respond to growth signals
o “Delayed Response” genes: CDKs and cyclins
♣ the CDKs and cyclins comprise heterodimers termed cyclin-dependent protein kinases (Kinases are enzymes that phosphorylate target proteins)
• The CDK subunit is the catalytic subunit, which phosphorylates the target (substrate)
o There are several different CDKs (CDKs 4, 2, and 1)
o During the cell-cycle, the content of each CDK remains constant
• The cyclic subunit is the regulatory subunit, which regulates the activity of the kinase heterodimer
o There are several cyclins (Cyclins D, E, A, and B)
o During the cell-cycle, the content of each cyclin increases
Clinical Correlation of Cell Cycle Checkpoints
o CLINICAL CORRELATION: Each of the proteins described to this point – Growth Factors, Growth Factor Receptors, early response genes, and CDKs/cyclins – are termed proto-oncogenes
♣ When a proto-oncogene is mutated, the protein it encodes becomes pathologically over-active stimulating the cell-cycle to induce cancer
Restriction Point (R)
• Regulation by extracellular growth factors is prominent in early G1, diminishing in late G1 before the restriction point (R)
o In response to growth factors, intracellular cyclins regulate discrete steps during interphase
♣ The content of cyclins D, E, A, and B increases during the cell-cycle in that sequence to activate the cell-cycle phases
• These increases occur concomitant with declining content of p27, a cell-cycle inhibitor
Cyclins DEAB
G1 Phase is activated by
- G1 Phase is activated by cyclinD/CDK4 heterodimer:
a. During early G1, growth factor activity induces sustained increase in cyclin D
i. A classical example is initiated by the Wnt growth factors, which induce increased content of the cytoplasmic protein -catenin, which migrates to the nucleus to activate transcription of the c-myc gene, which in turn activates the cyclin D gene
Wnt GF cytoplasmic -catenin –migrates to the nucleus c-myc gene cyclin D genei. Other factors also activate cyclin D transcription
1. GFs induce intracellular ras, a small GTPase-binding protein, which activates the MAP kinase pathway to effect cyclin D production
Cyclin D binds …
a. Cyclin D binds CDK4 to form cyclinD/CDK4 heterodimer
CyclinD/CDK4 phosphorylates …
a. CyclinD/CDK4 phosphorylates retinoblastoma (Rb) protein, forming P~Rb
i. In the phosphorylated state, Rb releases its hold on a transcription factor protein termed E2F
ii. Rb inhibits the cell-cycle; P~Rb releases this inhibition
E2F activates genes …
a. E2F activates genes encoding cyclin E and cyclin A, increasing their content
Basic Pattern of R regulation
CyclinD + CDK4 CyclinD/CDK4 phosphorylates Rb P~Rb releases E2F activates gene for cyclin E and cyclin A
S-Phase is activated by ….
- S-Phase is activated by cyclinE/CDK2 and cyclinA/CDK2 heterodimers
a. This depends on E2F-induced activation of genes encoding cyclins E and A
i. When content of cyclin E increases, the restriction checkpoint (R) regulated by cyclinE/CDK2 is breeched - Once breeched, the cell is committed to complete the cycle, all the way through G2 phase
- This commitment ensures that the whole genome is replicated during S-phase
- Failure to replicated all of the DNA results in cancer
b. During S phase, ‘DNA replication complexes’ which are ‘poised’ at multiple origins of DNA replication, are activated via phosphorylation of cyclinA/CDK2
i. This mechanism prevents formation of new, inappropriate DNA replication complexes
ii. This prevents the DNA from being replicated more than once
c. The end result of the cell-cycle is the precise apportionment of 1.0 genome to each daughter cell
Cyclin E restriction checkpoint is breeched cell is committed to complete cycle
CyclinA/CDK2 phosphorylates (and activates) “DNA replication complexes”
M-Phase is activated….
- M-Phase is activated at the G2/M boundary upon de-phosphorylation of the cyclinB/CDK1 heterodimer
a. This de-phosphorylation is mediated by a phosphatase termed cdc25
i. After it is de-phosphorylated, the cyclinB/CDK1 dimer enters the nucleus to phosphorlate many target proteins, causing: - Nuclear envelope breakdown (NEB)
- Mitotic spindle assembly
- Metaphase arrest
Anaphase regulation
Activated at mid-metaphase via the Anaphase Promoter Complex (APC)
Tumor Supressor Genes
• Activity of cyclin/CDK heterodimers is antagonized by proteins suppressing the cell-cycle
o Under the influence of such inhibitory proteins, cells are maintained in a prolonged G1-phase termed G0
♣ The genes that encode for these inhibitory suppressor proteins are termed tumor suppressor genes
♣ Mutation of a gene encoding a tumor suppressor protein can cause deactivation of that protein, resulting in cancer
o Retinoblastoma (Rb)
♣ Binds E2F-1, preventing E2F-1 from transcribing genes encoding cyclins E and A
♣ Rb is a “classic” tumor suppressor gene because when it is mutated, tumors of the retina termed “retinoblastomas” occur
♣ Rb, as well as proteins of related function such as p107 and p130, are termed “pocket proteins”
p21
o p21: Inhibits the cell-cycle by directly inhibiting CDK-2 and CDK-4
p27
o p27: similar to p21
p53
o p53 – the most prominent tumor suppressor
♣ Inhibits the cell-cycle via two functions
• (1) p53 induces transcription of the tumor suppressor p21
• (2) p53 promotes apoptosis (cell death)
♣ Is mutated in over ½ of human cancers
♣ Is referred to as the “sentinel of the genome,” since it induces killing of cells that contain inappropriately replicated DNA resultant from mistakes made during S-phase
Cancer (definition/cause)
Cancer: What is it?
• Cancer is a genetic disease displaying:
o Loss of cellular differentiation
o Increased proliferation and invasiveness of cells
o Changes in chromosomes: re-arrangement, loss, gain
Cancer: What causes it?
• Cancer is caused by inherited mutations and/or environmental insults to DNA during aging
o As we age, our cells evolve toward malignancy
o Cancer is definitely a genetic disease
Proto-Oncogenes
- Proto-oncogenes: about 70 genes that encode proteins that activate the cell cycle
a. Genes encoding cell surface receptors, factors, cyclins, and CDKs
b. When mutated, these are termed oncogenes
i. Mutated to be over-expressed, or the proteins the they encode have intensified activity
ii. Result is up-regulation
Tumor Supressing Genes
- Tumor Suppressing Genes: inhibit the cell-cycle
a. When mutated, products of these genes no longer “work”
b. About 20 tumor suppressors have been discovered, including:
Genes that Regulate Apoptosis
a. Diseased cells must be removed before they can colonize, so apoptosis is a normal and necessary mechanism
b. Stimulation of apoptosis causes tumors to regress
c. Apoptosis also regulates embryonic development, as shape (morphology) is attained – example: digit formation of fingers
a. Sequence of apoptosis:
i. Macrophages release TNF (tumor necrosis factor)
ii. TNF binds TNFR cell membrane receptor
iii. A balance between pro-apoptotic factors + anti-apoptotic factors is established
iv. Pro-apoptotic signals induce “leakiness” of the outer cell membrane of mitochondria
v. Cytochrome-C escapes to the cytoplasm
vi. Cytochrome-C activates Caspase, a protease that destroys chromatin, which fragments into 200 bp (i.e. nucleosomal) pieces
vii. Blebs appear on the cell surface and the cell defoliates (explodes)
Relationship of Apoptosis to Cancer
a. Relationship to cancer: In B cell lymphoma, a gene that normally inhibits apoptosis (bcl-2) is mutated
i. bcl-2 is an anti-apoptotic factor, but when mutated it is abnormally active
ii. Too much inhibition of apoptosis occurs, resulting in tumor formation
Telomerase
- Genes that induce cellular immortality: Telomerase
a. “Telomere hypothesis” – upon attaining a given length, the shortened telomere signals the cell to become senescent
i. This is a major regulatory mechanism in the aging process
b. In embryonic stem cells, and germ cells (sperm/egg), the telomere is restored to its original length after each cell division via an enzyme: telomerase
i. Telomerase is normally inhibited during aging of somatic cells so they normally become senescent after a critical number of cell divisions
ii. If the gene encoding telomerase is mutated, telomerase becomes active in somatic cells, rendering them immortal – this can cause carcinogenesis
Genes that repair DNA
a. Normally DNA damage causes the cell-cycle to stop until DNA repair enzymes fix the damaged DNA
b. If genes that encode DNA repair proteins are mutated, the proteins are inactivated, allowing clones of abnormal cells to arise
i. Recently documented in the instance of colo-rectal cancer
The Basic Properties of DNA Replication
• DNA synthesis is semiconservative
o Each daughter DNA molecule ends up with one of the original strands and one newly synthesized strand
• DNA is synthesized in the 5’ 3’ direction
• A 3’-OH primer and a template are required
o DNA polymerase I activity requires a single unpaired strand to act as a template and a primer strand to provide a free hydroxyl group at the 3’ end, to which a new nucleotide unit is added
o The catalytic mechanism of elongation likely involves two Mg2+ ions, coordinated to the phosphate groups of the incoming nucleotide triphosphate and to three Asp residues, two of which are highly conserved in all DNA polymerases
• DNA replication is semi-discontinuous
o There is a leading strand and a lagging strand
♣ The leading strand is transcribed in the direction of movement of the replication fork
♣ The leading strand is transcribed in the opposite direction, away from the movement of the replication fork
• Still transcribed 5’ 3’, the lagging strand uses an RNA primer to provide the free 3’ OH
• It is transcribed in segments called Okazaki fragments
DNA Replication has 4 Basic Steps:
- Separation of the two complementary strands at an origin of replication (a specific place(s) in the genome)
- Formation of the replication fork (primers and Okazaki fragments)
- Chain Elongation
- Removal of the primers
Properties of E. coli DNA Polymerase I
• One protein, 3 catalytic activities o 5’ 3’ polymerase o 5’ 3’ exonuclease o 3’ 5’ exonuclease Exonuclease: Proofreading activity: removes incorrectly base paired nucleotides
Error correction by the 3’ 5’ exonuclease activity of DNA polymerase I
- Polymerase mispairs dC with dT, which impedes translocation of DNA polymerase I to the next site
- Polymerase repositions the mispaired 3’ terminus into the 3’ 5’ exonuclease site which is behind the polymerase activity when the enzyme is oriented in its movement along the DNA
- Exonuclease hydrolyzes the mispaired dC
- The 3’ terminus repositions back to the polymerase site
- Polymerase incorporates the correct nucleotide, dA
DNA replication in Prokaryotic vs Eukaryotic organisms
• Process of DNA replication is functionally conserved between pro and euk organisms
o Many of the proteins are different but they function in an analogous manner
o A major difference in the replication process is that in prokaryotic replication this is one origin of replication and in eukaryotic replication there are multiple
Proteins of the E. Coli Replisome and their function(s)
- SSB – binding to single-stranded DNA
- DnaB protein (helicase) – DNA unwinding; primosome constituent
- Primase (DnaG protein) – RNA primer synthesis; primosome constituent
- DNA polymerase III – New strand elongation
- DNA polymerase I – Filling of gaps; excision of primers
- DNA ligase – Ligation
- DNA gyrase (DNA topoisomerase II) – Supercoiling
Telomeres
• Structures at the ends of eukaryotic chromosomes
• Have tandem repeats usually of T1-4G1-4 (with A-C on opposing strand)
• Can be tens of thousands of bp long in mammals
• TG strand is longer than its complement, leaves a 3’-overhang of several hundred bases
• Telomerase is an RNA-dependent DNA polymerase (reverse transcriptase) that carries its own RNA template
o The internal template of RNA of telomerase binds t and basepairs with the TG primer of DNA
o Telomerase adds more T and G residues to the TG primer, then repositions the internal template RNA to allow the addition of more T and G residues that generate the TG strand of the telomere
o The complementary strand is synthesized by cellular DNA polymerases after priming by RNA primase
Major Sources of DNA Mutation:
• Mis-Incorporation of nucleotides during DNA replication
o Adenine tautomer base pairs like Guanine
• Inherent chemical instability of bases (NONEZYMATIC)
o Deamination of CU; 100 bp/day
o Hydrolysis of N-glycosidic bond – depurination; 5000 bp/day
• Environmental Mutagens
o Deaminating agents (CU; GX)
♣ Nitrous acid precursors
• Sodium Nitrite (NaNO2), Sodium Nitrate (NaNO3), Nitrosamine
• Ionizing Radiation
o UV light can cause pyrimidine dimers
Environmental Mutagens
o Deaminating agents (CU; GX)
♣ Nitrous acid precursors
• Sodium Nitrite (NaNO2), Sodium Nitrate (NaNO3), Nitrosamine
o Alkylating agents (adding a methyl group) – methylated G binds with T
♣ Nitrogen mustards
♣ Aflatoxin
♣ Benzo(a)pyrene
♣ AAF (N-2-acetyl-2-aminofluorene)
o Intercalating agents – lead to insertion or deletion of one or more base pairs, alter reading frames
♣ Frameshift mutations caused by flat aromatic molecules:
• Ethidium bromide, acridines
Cells have multiple DNA Repair Systems
- Mismatch Repair: Mismatches
- Base-Excision Repair: Remove abnormal/damaged bases
- Nucleotide-Excision Repair: Remove large structural damages (thymine dimers)
- Direct Repair: Systems that recognize and repair with specific damages Pyrimidine dimers (photolyase)
- Recombinational Repair: Uses sister chromatid to repair damaged region
DNA Methylation and Mismatch Repair
• Methylation of DNA strands can serve to distinguish parent (template) strands from newly synthesized strains
o The methylation occurs at the N6 of adenines in (5’)GATC sequences
♣ This sequence is a palindrome because present in opposite orientations on the two strands
o After a few minutes, the new strand is methylated and the two strands can no longer be distinguished
A model for early steps of methyl-directed mismatch repair:
o MutS recognizes the mismatch
o MutH recognizes the (5’)GATC
o MutS and MutL form a complex at the mismatch
o DNA is threaded through the complex such that the complex moves simultaneously in both directions along the DNA until it encounters a MutH protein bound at the hemimethylated GATC sequence
o MutH cleaves the unmethylated strand on the 5’ side of the G in this sequences
o A complex consisting of DNA helicase II, SSB, and one of several exonucleases then degrades the unmethylated DNA strand from that point toward the mismatch
♣ The exonuclease that is used depends on the location of the cleavage site relative to the mismatch
o
The resulting gap is filled in by DNA polymerase III, and the nick is sealed by DNA ligase (Not shown)
Base Excision Repair
Nucleotide (BASE) Excision Repair on human DNA
• Some proteins involved in humans excision repair identified in patients with xeroderma pigmentosum (XP)
• Pathway:
o An excinuclease binds to DNA at the site of a bulky lesion and cleaves the damaged DNA strand on either side of the lesion
o The DNA segment –of 13 nucleotides or 29 nucleotides- is removed with the aid of a helicase
o The gap is filled in by DNA polymerase
o The remaining nick is filled in with DNA ligase
Direct Repair
• Some forms of DNA damage can be enzymatically reversed – for example, the removal of alkyl groups at the O6 position of Guanine
o Alkylation of guanine occurs from environmental mutagens and by cancer chemotherapeutic drugs
o MGMT (O-6-methylguanine-DNA methyltransferase) is the enzyme that repairs O6 methyl guanine
♣ It works by direct removal of the alkyl group
o This enzyme can interfere with some forms of chemotherapy that rely on DNA damage by alkylation
Therefore, inhibition of MGMT can facilitate cancer chemotherapy
Recombinational Repair
• Some lesions of DNA, such as double-stranded breaks, double-stranded cross-links, and lesions in single stranded regions, cannot be repaired using information from the complementary strand – Information must come from a separate homologous strand
• These types of lesions result from ionizing radiation and oxidative reactions
o Repair of these types of lesions is mediated by homologous genetic recombination called recombinational repair
Xeroderma Pigmentosum (XP
• Xeroderma Pigmentosum (XP) – a rare skin disease
o Individuals are extremely sensitive to UV light and develop skin cancer which metastasize - Patients often die before age 30
o XP results from a defect in the nucleotide excision repair mechanism for thymine dimers
♣ Recessive disease – fewer than 1,000 cases known worldwide
♣ Wide range of symptoms
• Blindness and deafness
• Blistering or freckling on minimum sun exposure
• Developmental disabilities
• Dwarfism and hypergonadism
• Increased skin and eye cancers
• Mental Retardation
Diseases Associated with Chromosomal Breakage
• Werner’s Syndrome: premature aging (adult progeria) – due to defects in a DNA helicase
• Bloom Syndrome: small body size, sun sensitivity, hypo and hyper-pigmented lesions, immunodeficiency, cancer, diabetes, and lung diseases – due to abolition of Ligase I activity needed to complete DNA repair
• Fanconi’s anemia, Ataxia telangiectasia, and Gardner’s Syndrome
o All believed to be due to defects in ligase activity
• Breast Cancer: Some forms of breast cancer are due to defects in breast cancer susceptibility genes (BRCA Genes)
o BRCA genes participate in DNA repair
(primary structure)
• Proteins spontaneously fold based on their sequence of amino acids (primary structure)
o most hydrophobic (apolar) amino acids within the core
o most hydrophilic (polar) amino acids on the surface
Secondary structures
• Secondary structures involve H-bonds between peptide bond carbonyl (C=O) groups and peptide bond amide hydrogens (>N-H) form first
o -helices, parallel and anti-parallel -pleated sheets
Basic AA structure
• There are 20 different genetically coded amino acids found in proteins
o Each aa (except proline) has a carboxyl group (-COOH), an amino group (-NH3), and a distinctive side change (R group) bonded to the -carbon atom
Chiral AA
• If the -carbon of an amino acid is attached to four different chemical groups, then that carbon is chiral and the amino acid is optically active
o Glycine is the only amino acid without optical activity because its -carbon is attached to two Hydrogen atoms
o AAs (except glycine) exist in either of two mirror image forms (enantiomers) termed the D and L, which have identical chemical properties in all respects except their solutions cause polarized light to rotate in opposite directions
♣ All amino acids found in mammalian proteins are of the L-configuration
Aliphatic amino acids
o Aliphatic amino acids possess side chains comprised of saturated carbon backbones – the subtle differences in the side chains allow for tight packing within the protein interior (7 –> GAVLIMP) – Grab All Vaginas Like Its My Pussy ♣ Glycine ♣ Alanine ♣ Valine ♣ Leucine ♣ Isoleucine ♣ Methionine ♣ Proline
Aromatic amino acids
o Aromatic amino acids possess side chains derived from either benzene (Phe and Tyr) or indole (Trp); each contains a methylene spacer (-CH2-) between the aromatic ring and the -carbon, this minimizes steric repulsion between the ring and the polypeptide chain backbone (3 –> PTT)
♣ Phenylalanine
♣ Tyrosine
♣ Tryptophan
Uncharged polar amino acids
o Uncharged polar amino acids have side chains that do not ionize at physiological pH (Met is sometimes classified in this group because of the slightly polar nature of its sulfur-containing side chain) (5 –> STAGC) Stop Touching All Girls C*nts ♣ Serine ♣ Threonine ♣ Asparagine ♣ Glutamine ♣ Cysteine
Charged polar amino acid
o Charged polar amino acids have side chains that can carry either a positive charge or a negative charge; although formally classified in other groups, the side chains of Tyr and Cys can ionize with pKa values close to physiological pH (5 –> LAHAG) Like All Hoes, Ashley Gags ♣ Lysine + ♣ Arginine + ♣ Histidine + ♣ Apartate + ♣ Glutamate +
The pKa of a group …
• The pKa of a group is the pH at which it is 50% protonated and 50% unprotonated
o At pH values lower than the pKa, there is a higher concentration of H+ in solution and >50% of the side chain group is protonated
o At pH values higher than the pKa, the solution has fewers H+ and <50% of the group is protonated
pI
• At some intermediate pH, the amino acid, peptide, or protein will have a net charge of zero; this pH is termed the isoelectric point, or pI
Sulfur-containing amino acids
• Methionine and Cysteine, the only amino acids containing sulfur (S), have unique roles and sensitivities
Significance of Met
o In newly synthesized (nascent) proteins, Met is always the first or N-terminal amino acid, but it also occurs at various positions throughout a polypeptide
o Under some conditions, the S of Met is oxidized, impairing protein function
Significance of Cys
• The thiol or sulfhydroyl group of cysteine can spontaneously oxidize to form a disulfide linking two cysteine residues within a protein
o Disulfide bonds form between specific cysteine residues because chain folding places the –SH groups in close proximity
♣ Disulfides rarely occur in intracellular proteins, but are common in extracellular proteins where their presence increases conformational stability of the structure
♣ Disulfide bonds are important in stabilizing the circulating peptide hormone insulin
Metamorphic proteins
• Metamorphic proteins exist in an ensemble of distinct structures of approximately equal energy that are in equilibrium (envision a thermodynamic folding funnel with two wells of similar depth)
alpha-Keratin
• -Keratin – a primary component of hair and nails
o Consists of two right-handed -helices that are intertwined in a left-handed supercoil called an -coiled coil
♣ Bonding between coils is both non-covalent (hydrophobic interactions, ionic bonds, H-bonds) and covalent (disulfide bonds between adjacent cysteine residues)
♣ Moderate number of disulfide bonds allow hair to stretch but then return to its original shape
• A much higher number of disulfide cross-links makes nails (and horns, claws, and hooves) rigid
♣ Belongs to a large family of coiled coil proteins that also includes the intermediate filaments that provide internal scaffolding for cells and myosin and tropomysin, which are involved in muscle action
Collagen
• Collagen – the most abundant protein in the body (20-25% of total protein) and exists as a superfamily of molecules whose type and organization are dictated by the structural roles each plays in different organs
o Typical collagen molecule is a long, rigid structure in which three polypeptides are wound around each other in a rope-like triple helix
o The individual collagen chains ( chains) are left-handed helices (NOT left-handed helices)
♣ 3 of them are wound around each other in a right-handed triple helix
♣ Multiple triple helices interdigitate to make collagen microfibrils
Clinical significance of Pro and Gly in Collagen
o Hydroxyproline and hydroxylysine are formed through post-translational hydroxylation reactions of proline and lysine and serve to stabilize the triple-helical structure
o The enzymes that catalyze the hydroxylation reactions require ascorbate (Vit C), a deficiency of which results in scurvy
♣ The symptoms of scurvy (e.g., bleeding gums) are due in part to the decreased tensile strength of collagen
Synthesis of Collagen
o Synthesis, post-translational modification, and triple helix assembly occur inside the cell, then the resulting procollagen molecule is secreted into the extracellular matrix where specific peptidases cleave N- and C- terminal propeptides
♣ Once removed, the mature collagen triple helix (tropocollagen) self-assembles into fibrils, with subsequent cross-linking to form mature collagen fibers
Elastin
• Elastin – A connective tissue protein with rubber-like properties that is found in the lings, large arterial walls, and elastic ligaments
o Comprised predominately of small, non-polar amino acids (glycine, alanine, valine) and is also rich in proline and lysine – contains few few hydroxyl derivatives
o Elastin is synthesized from a precursor, tropoelastin, which is secreted into the extracellular space where it interacts with specific glycoprotein microfibrils
♣ Some lysyl side chains are oxidized to form allysine residues which cross-link with lysine amino groups of neighboring polypeptides to produce elastin
Role of alpha-antitrypsin (1-AT) in elastin degredation
o Role of -antitrypsin (1-AT) in elastin degredation
♣ 1-AT is a small protein that inhibits a number of proteolytic enymes in bodily fluids
♣ Crucial function of 1-AT is to inhibit elastase, which is released by neutrophils and functions to degrade elastin
• Elastase destroys the alveolar epithelium if unregulated by 1-AT
• There are both genetic and environmental causes of insufficient functional 1-AT and both can lead to emphysema
o Genetic: 1-AT deficiency is predominantly due to the inheritance of two prominent mutant alleles, Z and S
♣ The Z allele causes a more severe deficiency and is due to an E342K substitution that causes 1-AT to be retained inside the cell
o Environmental: 1-AT contains a methionine residue that is essential for binding to target proteins (i.e., elastase)
♣ Elements in cigarette smoke cause the oxidation of the Met, which renders 1-AT unable to bind and inactivate elastase
♣ Heavy smokers can develop emphysema even if they have no mutant 1-AT alleles
Hemoglobin
• Adult hemoglobin (Hb) is a tetramer of two and two subunits
o Each subunit contains a heme cofactor to which O2 can be reversibly bound on the Fe2+ atom
o Hemoglobins of other subunit compositions occur developmentally
♣ Most important: Fetal Hb (HbF): comprised of 22
• O2 binding to Hb is cooperative such that binding of each O2 molecule to the tetramer increases the affinity with which the next O2 binds
o Similarly, dissociation of each O2 decreases affinity for those remaining so they dissociate more easily
o This cooperativity results in a sigmoidal O2 binding curve and that greatly increases the ability of Hb to release O2 in tissues
The affinity of Hb for O2 is decreased by:
o (1) High concentrations of H+ (lower pH)
o (2) CO2
o (3) 2,3-diphosphoglycerate (DPG)
o (4) Increasing Temperature (Fever)
Sickle Cell Disease `
• Sickle Cell Disease is an inherited mutation of the - globin gene causing a change from Glu to Val at position 6
o Presence of Val at position 6 allows deoxyHbS to polymerize into microfibrils that distort RBC shape and cause both hemolysis and vaso-occlusive disease
The Globin Genes
• On both chromosome 16 (alpha-globin family) and chromosome 11 (beta-globin family), the genes are expressed developmentally from 5’ 3’
• Expression of embryonic alpha-globin (zeta) occurs only briefly, and then the adult form (alpha) dominates through fetal growth and into adulthood
• Each chromosome 16 has 2 alpha-globin genes (a person has 4 total); each is active and codes about ¼ of expressed alpha-globin
o Consequently, a defect in 1 or 2 alpha-gene clusters is not too serious
• Expression of embryonic beta-globin (epsilon) is also brief but followed by a fetal form (gamma) that does not disappear until after birth
o HbF is clinically important
• There are two forms of adult Hb: HbA and HbA2
o HbA2 usually accounts for only a few present of adult Hb
• Each chromosome 11 has only one functional beta-globin gene
o Beta-globin gene defects are more likely to have clinical consequences
Overview of gas transport and pH regulation in humans
• Hemoglobin is a specialized protein designed to transport oxygen from the lungs to the peripheral tissues where oxygen tension is low
o Metabolism in the peripheral tissues generates CO2 and H+ that are transported back to the lungs, in part by hemoglobin
O2 solubility in plasma
• O2 has very low solubility in plasma
o >98% of the O2 that reaches tissues is carried in RBCs bound to Hb
o Situation is different for CO2
♣ RBCs contain carbonic anhydrase, which catalyzes the rapid reversible hydration of CO2 to carbonic acid (H2CO3)
• H2CO3 then rapidly and spontaneously dissociates to bicarbonate (HCO3-) and a H+
• CO2 and HCO3- are soluble in plasma and RBC cytosol and most of the CO2 made in tissues returns to the lungs as those species
o About 14% of the CO2 made is carried bound to Hb
Tertiary Structures of Myoglobin and Hemoglobin
• Hemoglobin is a heterotetrameric protein having the subunit composition ()2
o The alpha and beta subunits have similar sequences and tertiary structures
o Both subunits are evolutionarily related to myoglobin, a monomeric protein abundant in muscle that is designed to store O2
♣ Both proteins contain a Fe2+-protoporphryin IX prosthetic group that is responsible for binding O2
• Note: Fe2+ is the ferrous form of iron that is capable of binding O2
• Fe3+ is the ferric form that cannot bind O2, and is present in an inactive form of hemoglobin called methemoglobin
Structure-function relationships in myoglobin and hemoglobin
• The partial pressure of dissolved oxygen in aqueous solution is proportional to the partial pressure in the gas phase
o Myoglobin gives a normal binding curve, which is hyperbolic in shape
o Hemoglobin shows sigmoidal cooperative binding of the oxygen that is a direct result of its more complex subunit structure
How O2 binding changes the conformation of the Hb subunit
• Without O2 bound, the heme Fe2+is pulled away from the plane of the porphyrin ring by a His residue of the polypeptide chain
o When O2 binds, it pulls the Fe2+ back into the plane of the ring and that moves the His residue and its whole section of the polypeptide chain
o That in turn causes the Hb subunits to shift relative to one another to an arrangement that favors the R-conformation
Bohr effect
o The reciprocal relationship between O2 and H+ is termed the Bohr effect, or isohydric shift
o Changes in H+ binding result from a shift in the pKa of specific residues (mostly histidines) due to charges microenvironment effects trigged by conformational changes in the hemoglobin molecule
2,3-diphosphoglycerate (2,3-DPG or DPG)
• DPG is a negative allosteric effector of O2 binding
o DPG binds to a specific site in a cleft between the beta subunits
♣ Binding stabilized mostly by ionic interactions
o Special regulatory mechanisms exist in RBCs to control the concentration of 2,3 DPG in order to fine tune the affinity of hemoglobin for O2 in response to changes in metabolism and environment
♣ This obviates the need to induce the synthesis of different hemoglobin isoforms of altered O2 affinities (which is not possible for human RBCs since they lose the capacity to synthesize protein during their terminal differentiation)
Ribonucleic acids play three well-understood roles in living cells:
- Messenger RNAs: encode amino acid sequences of the polypeptides found in the cell (5% of total RNA, most complex)
- Transfer RNAs: match specific amino acids to triplet codons in mRNA during protein syn (~15% of total RNA (and snRNA in eukaryotes)
- Ribosomal RNAs: the constituents and catalytic appropriate amino acids (~80% of total RNA, least complex)
Ribonucleic acids play several less-understood functions in eukaryotic cells
o MicroRNA: appears to regulate the expression of genes, possibly via binding to specific nucleotide sequences
o Other functions: Ribonucleic acids act as genomic material in viruses
RNA Metabolism
• Ribonucleic acids are synthesized in cells using DNA as a template in transcription
o Transcription is tightly regulated in order to control the concentration of each protein
• Being mainly single-stranded, many RNA molecules can fold into compact structures with specific functions
o Some RNA molecules can act as catalysts (ribozymes), often using metal ions as cofactors
• Most Eukaryotic ribonucleic acids are processed after synthesis
o Elimination of introns; joining of exons
o Poly-adenylation of the 3’ end
o Capping the 5’ end
Transcription in Prokaryotes
• Basic Reaction:
o Nucleoside triphosphates added to the 3’ end of the growing RNA strand
o Chemically the RNA polymerization reaction is very similar to DNA synthesis, occurring in the 5’ to 3’ direction
o The 3’ hydroxyl group of the existing RNA chain undergoes a nucleophilic attack on the alpha-phosphate of the incoming nucleotide
o The growing chain is complementary to the template strand in DNA
o The synthesis is catalyzed by an enzyme (RNA polymerase)
♣ Two Mg2+ ions are used as cofactors among many Asp residues
Basic Properties of RNA Polymerases
• In prokaryotes, a single RNA polymerase synthesizes mRNA, rRNA, and tRNA
• Prokaryotic RNA polymerase is a multisubunit enzyme
o RNA polymerase holoenzyme has five core subunits of 2’ plus a sixth,
o The core enzyme is responsible for polymerization but it lacks specificity and cannot recognize the promoter
♣ The “sigma” factor allows the holoenzyme to recognize promoter regions on the DNA
o RNA pol lacks 3’ 5’ exonuclease, so it has a high error rate of 1/104-1/105
♣ You don’t have to worry about inheriting them because they are largely unstable, so it doesn’t matter
o RNA binds to promoter regions to initiate transcription
Anatomy of a bacterial gene
- Promoter: site for binding RNA polymerase
- Operator: binding sites for repressor of activator
- Structural gene: often times many related genes transcribed as a single unit
- Together they are called an operon
Initiation of transcription requires several steps generally divided into two phases:
o (1) Binding phase – the initial interaction of the RNA polymerase with the promoter leads to formation of a closed complex, in which the promoter DNA is stable bound but not unwound
♣ The sigma subunit helps provide specificity
♣ A 12 to 15 bp region of DNA is then unwound to form an open complex
o
(2) Initiation phase – encompasses transcription initiation and promoter clearance
Elongation
• Elongation – once commenced, the sigma subunit is released and it replaced by the protein NusA
o The polymerase leaves the promoter and becomes committed to elongation of the RNA
Termination
• Termination – once transcription is complete, the RNA is released, the NusA protein dissociates, and the RNA polymerase dissociates from the DNA
DNA Template Strand:
• DNA Template Strand: serves as template for RNA polymerase
DNA Coding Strand:
• DNA Coding Strand: the non-template strand; has the same sequence as the RNA transcript
The Sigma Subunit of the Holoenzyme
The Sigma Subunit of the Holoenzyme Enables RNA Polymerase to Recognize Promoters – Not All Promoters Are Created Equal
• Different sigma factors recognize different promoters
• Sigma increases specificity of RNAP, but decreases affinity
• Strong promoters cause frequent initiation and tend to conform closely to the consensus
• Different sigma factors lead to differential gene expression: Change in sigma utilization can be used to regulate developmental changes in gene expression
Transcriptional Elongation
• Elongation proceeds in the 5’ to 3’ direction partially through the energy released by cleavage of the phosphate bond in the incoming ribonucleotide, and by subsequent hydrolysis of pyrophosphate to inorganic phosphate
o The sigma factor dissociates from the holoenzyme complex immediately after elongation begins
• Multiple RNA polymerase complexes load onto the promoter region in sequential fashion, allowing the gene to be transcribed continuously
o The rate of elongation by E. coli polymerase is about 30 nt/second
o Elongation is extremely processive
♣ Once sigma dissociates, a single RNAP can synthesize thousands of nucleotides before it dissociates
transcription bubble
• A transcription bubble forms during the process of elongation
o Negative superhelicity in the double-stranded DNA facilitates the melting of the two strands of DNA and the formation of this bubble
o The RNA transcript is only base paired with the DNA template in this region, leaving a growing chain behind as the DNA helix reforms behind the elongation complex
