Final Exam - Chapters 7, 26, 27, and 28

RNA capping

5' end of RNA with triphosphate group, pppNp + phosphohydrolase -(-Pi)> ppNp + GTP, Gppp + guanylyltransferase -(+PPi)> GpppNp + adoMet + guanine-7-methyltransferase -(adoHcy)> M^7GpppmNp, 5' end of RNA with cap cap-synthesizing complex CBC - cap binding complex cap reminds bound to CTD via CBD

formation of the transcript and its processing during maturation of mRNA

1. 5'-capping - added before transcript is complete 2. splicing 3. 3' polyadenylation may occur before or after splicing - poly(A) tail occurs in the nucleus transcription and 5' capping -> completion of primary transcript -> cleavage, polyadenylation, and splicing (DNA -> primary transcript -> mature mRNA) RNA released from DNA when polymerase II is cleaved

thousands of nucleic acid analogs of RNA and DNA have been made and tested in antisense oligonucleotides: FDA approved antisense drugs

1. Fomivirsen 2. Mipomersen - ~$176,000/year 3. Nusinersen - ~$375,000/year 4. Eterplirsen - ~$300,000/year chemistry of RNA interference - chemical modification of DNA to make it more stable and easier to deliver oligonucleotide drugs - very expensive

reverse transcriptases catalyze 3 reactions

1. RNA-dependent DNA synthesis 2. RNA degradation 3. DNA-dependent DNA synthesis contain Zn2+, like DNA polymerase use a primer of tRNA lack 3' -> 5'-proofreading, like RNA polymerase - make reverse transcriptase error-prone this explains the high rate of virus mutation/evolution and drug resistance degrade rna - nuclease activity evolve much faster than other viruses promoting tumors - increases cell replication

prokaryotic ribosome

70S - 50S and 30S subunits

eukaryotic ribosome

80S - 60S and 40S subunits

protein synthesis involves five stages

1. activation of amino acids - tRNA is aminoacylated with corresponding aa 2. initiation of translation - recognizing the AUG codon; mRNA and aminoacylated tRNA bind to the small ribosomal subunit (the large subunit then binds as well) 3. elongation - successive cycles of aminoacyl-tRNA binding and peptide bond formation occur until a STOP codon is reached 4. termination and ribosome recycling - translation stops when a stop codon is encountered, the mRNA and protein dissociate, ribosomal subunits recycled 5. folding and post-translational processing - catalyzed by a variety of enzymes

two models for gene expression in prokaryotes

1. inducible system 2. repressible system

RNA processing

1. mRNA capping - 5' cap 2. mRNA polyadenylation 3. mRNA splicing 4. regulation of mRNA splicing 5. exploit the RNA splicing to treat spinal muscular atrophy (SMA) restricted to eukaryotic sites; minimal in prokaryotes

amino-terminal sequences of some eukaryotic proteins that direct their translocation into the ER

1. mRNA is scanned to find AUG codon 2. signal sequence is found - signal recognition particle (SRP) (signal sequence is a short amino acid sequence) 3. stops at 70 amino acids in the cytosol - ribosome receptor on ER lumen (peptide translocation complex and SRP receptor) 4. GTP introduced and protein is "spit" into the ER lumen - signal peptidase 5. GDP + Pi released and SRP is recycled 6. protein continues to be synthesized and goes into ER lumen 7. signal sequence is removed 8. ribosomal subunits dissociate and recycled

deoxyribonucleic acid (DNA)

1. sugar - deoxyribose 2. structure - two strands, arranged in a double helix 3. bases - Adenine ('A'), Guanine ('G') and Cytosine ('C'), and Thymine ('T') 4. stability - more stable deoxyribonucleotide - H on the 2' C

ribonucleic acid (RNA)

1. sugar - ribose 2. structure - one strand, sometimes forms a secondary double helix structure 3. bases - Adenine ('A'), Guanine ('G'), Cytosine ('C'), and Uracil ('U') 4. stability - less stable; -OH group exposed to attack ribonucleotide - -OH on the 2' C

essential components for step 1 of protein synthesis - activation of amino acids

20 amino acids 20 aminoacyl-tRNA synthetases 32 or more tRNAs ATP Mg2+

PXR ligands - prescriptions

Cyclophosphamide - immunosuppressive, antineoplastic Cyproterone acetate - antineoplastic, contraceptive Taxol - anticarcinogenic Tamoxifen - anticarcinogenic Spironolactone - diuretics Dexamethasone - anti-inflammatory Troglitazone - hypoglycemic Nifedipine - vasodilator Mifepristone - abortifacient, contraceptives Rifampicin - antitubercular (anti-TB drug) Clotrimazole - antifungal Ritonavir - antiretroviral Phenobarbital - anticonvulsants, hypnotics, and sedatives Glutethimide - hypnotics Efavirenz - antiretroviral

some common monosaccharides

D-ribose D-deoxyribose D-glucose D-mannose D-galactose D-fructose all sugars merge into the main pathway of degradation -glycolysis! all of these are consumed in the diet and degraded by glycolysis - covered to glucose or another intermediate of the pathway in order to be degraded by the process and liberate energy

information pathways

DNA -> RNA -> protein

consensus sequence of many E. coli promoters

DNA 5' -> UP element -> TTGACA (-35 region) -> N17 -> TATAAT (-10 region) -> N5-9 -> RNA start site -> mRNA

key concepts of transcription - coding strand and direction of transcription

DNA coding strand is identical to the mRNA - except T for U DNA template strand is complementary and antiparallel to the mRNA - RNA sequence similar to coding strand (T versus U) direction of transcription - 5' -> 3' (DNA -> mRNA); RNA polymerase travels along the template strand in the 3′ → 5′ direction, which allows for the construction of transcribed mRNA in the 5′ → 3′direction direction of translation - 5' -> 3' (mRNA -> protein) growing end of new RNA temporarily base-pairs with DNA template for ~8 bp coding information may be on either strand of DNA

DNA methylation in promoter region silences gene expression

DNA methylation (5-methylcytosine) at promoter CpG sites leads to transcription repression the methylation involves DNMT methyltransferase - transfers a methyl group to CpG island in the promoter region of DNA, leading to gene silence hypermethylation of tumor suppressor genes leads to cancer 5-azacytidine (Vidaza) inhibit DNMT methyltransferase - cancer drugs increase demethylation of promoter regions and tumor death methylated genes cannot be expressed

enhancers

DNA sequence outside the normal promoter regions can be recognized by specific transcription factors to enhance transcription levels DNA often must bend into a hairpin loop to bring these elements together spatially 100 bp - promoter region enhancers are farther away from the promoter region - close to region upon bending on DNA (recruit RNA polymerase II to begin translation)

flow of genetic information from DNA to protein

DNA to mRNA by transcription mRNA to protein by translation DNA coding strand is identical to the mRNA except T for U DNA template strand is complementary and antiparallel to the mRNA direction of transcription and translation in 5' -> 3' direction RNA polymerase binds to the template strand to synthesize mRNA

E, P, and A sites - initiation in eukaryotes

E = exit site P = peptidyl site - where the bond forms between amino acids A = acceptor site order of sites in the ribosome during translation - APE 1. start of translation 2. elF4F binds cap site - recognizing the 5' cap by elF4F and brings in the small subunit of ribosome 5'-UTR - untranslated region 3. scanning - possible codons to find AUG 4. mRNA AUG codon is reached 5. 60S subunit enters and next codon is read - recruiting larger subunit of ribosome to extend peptides

class I versus class II

E. coli Gln-tRNA synthetase - class I yeast dimeric Asp-tRNA synthetase - class II short RNA mini-helix, with the critical G=U bp but lacking most of the remaining tRNA structure this is aminoacylated specifically with Ala almost as well as the full tRNA(Ala)

ubiquitin-dependent protein degradation

E1 (plus ATP) first adenylates the carboxy-terminal carboxylate of ubiquitin (Ub), forming UbAMP, and then forms a Ub thioester intermediate (E1-Ub) ubiquitin is transferred from E1 to E2, and then to the protein target with assistance from E3 typically, an isopeptide bond is formed between the ubiquitin C-terminal carboxylate and the ɛ-amino group of a Lys side chain of the substrate protein or the growing ubiquitin chain ubiquitin - 76 amino acids; labels protein to be degraded the 26S proteasome is highly conserved in all eukaryotes the two subassemblies are the 20S core particle and the 19S regulatory particle a regulatory particle forms a cap on each end of the core particle - particles recognize ubiquinated proteins the regulatory particle unfolds ubiquitinated proteins and translocates them into the core particle degrades proteins to 3-25 aa's - digested to single amino acids available for recycling eukaryotic proteasome - substrate protein, polyubiquitin attached to protein interacts with proteasome, 19S regulatory particle

regulation of chromatin structure - Euchromatin

Euchromatin is looser and appears light under the microscope the transcription machinery can access the genes of interest, so these genes are active more accessible to be translated - RNA polymerase II and transcription factors

PXR ligands - herbal medicines

Glycyrrhiza uralensis Fisch Schisandra chinensis Baill Kava St. John's wort - antidepressant herbal various fungi Taxus brevifolia Coleus forskohlii Sutherlandia frutescens

regulation of chromatin structure - Heterochromatin

Heterochromatin is tightly coiled DNA that appears dark under the microscope its tight coiling makes it inaccessible to the transcription machinery, so these genes are inactive remodeling of the chromatin structures regulates gene expression levels in the cell

transcription

Inr = initiator sequence 1. polymerase II is recruited to the DNA by transcription factors - preinitiation complex closed 2. the transcription bubble forms - initiation complex opens and transcription initiates 3. the CTD is phosphorylated during initiation - the polymerase escapes the promoter and elongation factors introduced 4. transcription elongation is aided by elongation factors after TFIIE and TFIIH dissociate 5. elongation factors dissociate - the CTD is dephosphorlyated as transcription terminates, a process facilitated by transcription factors (termination complex)

structure and gene retroviral products

LTR regulate transcription in host cell DNA packaging transcription forms the primary transcript translation forms: 1. polyprotein A - proteolytic cleavage -> integrase, protease, reverse transcriptase, and virus structural proteins 2. polyprotein B - proteolytic cleavage -> viral envelope proteins

a single gene can yield different products depending on RNA processing

RNA can be "edited" - bases removed/added/changed cleavage/polyadenylation patterns can vary, yielding different mature transcripts immunoglobulin heavy chain gene - different degrees of polyadenylation and different cleavage sites yield diverse sequences calcitonin and calcitonin-gene-related peptide, in rat thyroid and brain, respectively, made from same mRNA discrepancy between gene number and protein number modulated by RNA processing - many more proteins than genes

retroviral infection of a mammalian cell and integration into host chromosome

RNA genome inside retrovirus -> cytoplasm -> RNA -> reverse transcription -> viral DNA -> nucleus -> integration in the chromosome of the host cell transcription -> transport out of nucleus -> ribosome -> translation and proteolysis -> assembly and packaging -> lysis -> new retrovirus finding ligand to integrate in the cell

elF4A

RNA helicase activity removes secondary structure in the mRNA to permit binding to 40S subunit part of the elF4F complex

cellular mRNAs are degraded at different rates

RNA lifetime is one means of gene regulation half-lives vary from seconds to hours - typical vertebrate mRNA ~3 hrs ~10 turnovers per cell generation shorter (~1.5 mins) half-lives for bacterial mRNAs degradation via ribonucleases hairpin structures in mRNA can extend half-life protective structure against 5' or 3' end of ribonuclease can extend half life

eukaryotes contain several distinct polymerases

RNA polymerase I, II, and III mitochondria have their own RNA polymerase

three eukaryotic RNA polymerases versus three types of RNA

RNA polymerase: location; products, α-Amanitin sensitivity I: nucleolus; large rRNAs (28S, 18S, 5.8S); insensitive II: nucleus; pre-mRNA, some snRNAs; highly sensitive III: nucleus; tRNA, small rRNA (5S), snRNA; intermediate sensitivity

RNA polymerase binding to promoters is a major target of regulation

RNA polymerases bind to promoter sequences near or at distant sites from the starting point of transcription initiation the RNA pol-promoter interaction greatly influences the rate of transcription initiation regulatory proteins (transcription factors) work to enhance or inhibit this interaction between RNA polymerase and the promoter DNA upstream and downstream factors

PXR-mediated drug-drug interactions - example 2

St. John's wort + cyclosporine -> organ transplant failure cyclosporine is metabolized by CYP3A, which is induced by St. John's wort - drug interaction that increases metabolism of cyclosporine (less protection against rejection of transplanted organ)

transpeptidase mechanism

TP = transpeptidase enzyme that crosslinks the peptides two step mechanism of transpeptidase: 1. activation - active nucleophilic serine in the enzyme structure attacks the last alanine present in the pentapeptide and displaces it (enzyme now attached to the peptide, called the acyl-enzyme intermediate) 2. crosslinking - amino group side chain is nucleophilic and attacks the electrophilic carbonyl attached to the enzyme (enzyme is released and peptides are linked, forming cross linked peptide side chain)

gangliosides and diseases

Tay-Sachs disease Guillain-Barré syndrome influenza

how miRNAs are processed to prevent translation

a special class of dsRNAs involved in gene regulation that are ~22 nt long and complementary in sequence to particular regions of mRNA regulating mRNA function by cleaving the mRNA or suppressing its translation RNAi and miRNA - miRNA therapeutic potential MOA of miRNA Drosha & Dicer are RNases originally synthesized in nucleus - double stranded RNA not translated into protein - partial complementarity

stage 4 - termination

UAA, UAG, or UGA in the A site will trigger the action of termination factors (release factors) RF-1 (UAG & UAA), RF-2 (UGA), RF-3 (?) eRF binds to all 3 stop codons in eukaryotic cells these help to hydrolyze terminal peptide-tRNA bond release peptide and tRNA from ribosome and cause subunits of ribosome to dissociate so that initiation can begin again release factor binds -> polypeptidyl-tRNA link hydrolyzed -> components dissociate EF-G = elongation (translocation) factor RF = release factor - no tRNA matches the stop codons (binding of the release factor instead) IF = initiation factor RRF = ribosome recycling factor

immunoglobulin recombination and alternative splicing

V segments - 1 to ~300 J segments C segment - germ-line DNA recombination resulting in deletion of DNA between V and J segments - mature light-chain gene mature light chain gene - V84 + J4 (DNA of B lymphocyte) transcription forms the primary transcript removal of sequences between J4 and C by mRNA splicing forms processed mRNA translation forms the variable region and constant region of the light-chain polypeptide protein folding and assembly forms the antibody molecule with light and heavy chain recombination - diverse pool of antibodies in immune system different V, J, C combinations

5' cap

a 7-methylguanosine 7-methylguanosine links to the 5' end of the transcript via 5',5'-triphosphate linkage - protects from attack may include additional methylations at 2'-OH groups of then next two nucleotides methyl groups derive from S-adenosyl-methionine (SAM) protects RNA terminus from nucleases forms a binding site for ribosome - translation synthesis of the cap is carried out by enzymes tethered to the carboxyl terminal domain (CTD) of polymerase II

operon

a cluster of genes sharing a promoter and regulatory sequences genes are transcribed together - several genes represented on one mRNA polycistronic mRNA - multiple open reading frames

genetic code is degenerate

a given amino acid may be specified by more than one codon only methionine and tryptophan have single codons degenerate does not mean imperfect - the genetic code is unambiguous because no codon specifies more than one amino acid the degeneracy of the code is not uniform amino acids have anywhere from 1 to 6 number of codons - 61 total codons 61 codons for 20 or 21 amino acids - 3 stop codons

Tay-Sachs disease

a lipid storage disease caused by absence of the enzyme hexosaminidase, which functions in ganglioside metabolism deficiency of the enzyme results in accumulation of gangliosides in the cells of the brain, causing neurological deterioration there is no cure for Tay-Sachs disease, but some treatments can help in managing symptoms - respiratory care, physical therapy, etc.

glucometers

a medical device for determining the approximate concentration of glucose in the blood glucometers use test strips containing glucose oxidase, an enzyme that reacts to glucose in the blood droplet, and an interface to an electrode inside the meter when the strip is inserted into the meter, the flux of the glucose reaction generates an electrical signal the glucometer is calibrated so the number appearing in its digital readout corresponds to the strength of the electrical current the more glucose in the sample, the higher the number

trehalose

a non reducing sugar formed by α,α-1,1-glycosidic bond is the major constituent of the circulating fluid in insects two alpha-D-glucose monosaccharides - condensation reaction

isomerism

a phenomenon in which compounds have the same molecular formula but exist in different forms owing to different arrangement of atoms

proteoglycans - glycoproteins performing structural functions: aggrecan

a proteoglycan that is a key component in the extracellular matrix of cartilage - higher percentage of carbs than proteins also known as cartilage-specific proteoglycan core protein (CSPCP), or chondroitin sulfate proteoglycan 1 this is a protein that, in humans, is encoded by the ACAN gene the encoded protein is an integral part of the extracellular matrix in cartilagenous tissue and it withstands compression in cartilage - serves as shock absorber the protein component of ACAN consists of 2397 amino acids with three globular domains the site of glycosaminoglycan attachment is the extended region between globular domains 2 and 3 the glycoaminoglycans attached are usually keratin and chondroitin sulfate several ACAN's are noncovalently bound to a long filament through their first globular domain the long filament is formed by linking glycosaminoglycans hyaluronan water is bound to the glycosaminoalycans because of which aggrecans can cushion compressive forces

antibodies

are part of the immune system are Y-shaped proteins bind to the antigens on the red blood cells this leads to clumping and destruction of the cells

summary of stage 4 - termination

after many such elongation cycles, synthesis of the polypeptide chain is terminated with the aid of release factors

chemical basis for ring formation

aldehyde + alcohol <-> hemiacetal ketone + alcohol <-> hemiketal lone pair on oxygen attacks carbonyl, opening up the double bond - structures are interconvertible and are in equilibrium in solution carbs - intramolecular formation where the hydroxy group on the chiral carbon is farthest from the carbonyl group (achiral carbonyl group becomes chiral when introduced into ring structure - anomeric carbon becomes alpha or beta) these molecules have directionality - starts with non-reducing end and ends with the reducing end

monosaccharides - reducing agents

aldoses (glucose) act as reducing sugars because the aldehyde group in monosaccharides can react with the oxidizing agent (Cu2+) and reduce it example - beta-D-glucose <-> D-glucose + Cu2+ <-(H2O, Ho-)-> Cu+ (Cu2O) + gluconic acid ketoses (fructose) act as reducing sugar as they can tautomerize to aldoses, which then reacts with oxidizing agents to reduce it example - fructose <-> glucose and mannose - the aldehyde group in glucose and mannose react with oxidizing agents and reduce them the sugar gets oxidized in the process - aldehyde oxidized to carboxylic acid and reduces the oxidizing agent, and ketones cannot be oxidized! (must tautomerize)

glycolysis

all carbohydrates consumed in the diet enter the key metabolic pathway called glycolysis to harvest energy, thus acting as the body's primary source of energy

potential reading frames in the genetic code

all mRNAs have three potential reading frames the triplets, and hence the amino acids specified, are different in each reading frame the code is written in the 5 → 3' direction third base (wobble site) is less important in binding of tRNA first codon establishes the reading frame - if reading frame is thrown off by a base or two, all subsequent codons are out of order (frame shift mutation) translational frame shifting and RNA editing can affect how the genetic code is read

repressible system

allow constant production of a protein product the repressor made by the regulator gene is inactive until it binds to a corepressor the corepressor is usually the end product of the metabolism this complex then binds the operator site to prevent further transcription repressible systems tend to serve as negative feedback

wobble position of tRNA

allows some tRNAs to recognize more than one codon - 1 anticodon recognizing up to three different codons inosinate can form hydrogen bonds with three different nucleotides - U, C, and A these pairings are rather weak compared with the strong hydrogen bonds - G≡C and A=U adenosine + H2O - NH3 -> inosine anticodon - 3'-G-C-I-5' codons: 1. 5'-C-G-A-3' 2. 5'-C-G-U-3' 3. 5'-C-G-C-3' H-bonding of Inosine to C, G and U as wobble base wobble position in tRNA anticodons allows some tRNAs to recognize more than one codon

aminoacylation of tRNA by aminoacyl-tRNA synthetases

alpha-carboxyl of amino acid attacks alpha-phosphate of ATP, forming 5'-aminoacyl adenylate (aminoacyl-AMP) - lose PPi linking amino acid to ATP -> aminoacyl AMP - two pathways from here: 1. aminoacyl group is transferred to 2'-OH of the 3'-terminal A residue of tRNA, releasing AMP - transesterification moves aminoacyl group to 3'-OH of the same tRNA residue, generating the aminoacyl-tRNA product CCA attacking this complex, forming free AMP and aminoacyl-tRNA - bound to third position (A) 2. aminoacyl group is transferred directly to the 3'-OH of the 3'-terminal A residue of tRNA, generating the aminoacyl-tRNA product

carbohydrates

also called sugars - polyhydroxy aldehydes or ketones or substances that yield these compounds on hydrolysis examples - D-ribose, D-glucose, and D-fructose (several glucose molecules stored as glycogen) heterogeneous molecules - large in size essential biomolecules are the body's primary source of energy (1 g of carbohydrate contains ~4 Kcal of energy) and also as energy storage molecules are components of nucleic acids (DNA and RNA) components of cell wall - cell wall of plants and bacteria are made up carbohydrates that function in protection, shape, transport, communication, etc. linear versus cyclic - prefer cyclic form in solution D or L isomers based on position of hydroxy groups - looking at the -OH group farthest from the carbonyl carbon on the chiral carbon (D = -OH on right, L = -OH on left)

biochemical properties of monosaccharides

altered by reaction with other molecules - altering functional groups increases diversity beta-L-fucose (Fuc) - component of glycoproteins and glycolipids beta-D-acetylgalactosamine (GalNAc) beta-D-acetylglucosamine (GlcNAc) - part of structural polymers, including bacterial cell wall sialic acid (Sia; N-acetylneuraminate) - occurs in glycoproteins and glycolipids on animal surfaces providing sites of recognition by other cells or extracellular carbohydrate binding proteins -OH group changed to metal, amino, or acylated (examples above) glucose 6-phosphate (G-6P), dihydroxyacetone phosphate (DHAP), glyceraldehye 3-phosphate (GAP) - key intermediates in energy generation and biosynthesis phosphorylated monosaccharides - energy generation; G-6P is the first molecule to enter the glycolysis pathway beta-D-glucuronate, D-glucuronate, and D-glucono-gamma-lactone - oxidized forms, counter ions when administering cations (hold a negative charge) these are all examples of modified saccharides with increased biochemical versatility

aminoacyl-tRNA general structure

amino acid arm - 3' end of tRNA (NH2-CR'H-CONH-; NH2-CHR-CONH-CR'H-CONH-) TC arm extra arm anticodon arm -> anticodon D arm ribosome does not "care" what aa is on the 3'- terminus of the t-RNA

summary of stage 1 of protein synthesis

amino acids are activated by specific aminoacyl-tRNA synthetases in the cytosol - very important enzyme these enzymes catalyze the formation of aminoacyl-tRNAs, with simultaneous cleavage of ATP to AMP and PPi the fidelity of protein synthesis depends to a large extent on the accuracy of this reaction, and some of these enzymes carry out proofreading steps at separate active sites - making sure the right amino acid is linked to the correct tRNA

deletion mutation

an amino acid within the sequence is deleted alters the reading frame

heparan sulfate

an ubiquitous cell surface component, as well as extracellular substance in blood vessel walls and the brain also has anticoagulant property, but lower than heparin

tetracycline

antibacterial drug inhibits elongation by blocking the attachment of charged aminoacyl-tRNA to the A site on the bacterial ribosome

streptomycin

antibacterial drug inhibits protein synthesis by binding to the small 16S rRNA of the 30S subunit of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit

cycloheximide

antibacterial drug inhibits the translocation step in protein synthesis and stalls elongation in eukaryotic ribosome

chloramphenicol

antibacterial drug prevents protein chain elongation by inhibiting the peptidyl transferase activity of the bacterial ribosome - binds to A2451 and A2452 residues in the 23S rRNA of the 50S large ribosomal subunit, preventing peptide bond formation

puromycin

antibacterial drug puromycin resembles the aminoacyl end of a charged tRNA, and it binds to the ribosomal A site and participates in peptide bond formation the product of this reaction, peptidyl puromycin, is not translocated to the P site instead, it dissociates from the ribosome, causing premature chain termination

long non-coding RNA

appear to be involved in organizing nuclear organization and regulation of gene expression

microRNA

appears to regulate the expression of genes, possibly via binding to specific nucleotide sequences

proteoglycan

are proteins bonded to glycosaminoglycans - proteins that are heavily glycosylated occur in the extracellular matrix of the cell, functioning as structural components and lubricants have higher carbohydrate content in comparison to simple glycoproteins

transcription initiation

assembly of RNA polymerase II at promoter initiated by TATA-binding protein (TBP) with the promoter - TBP is part of a multisubunit complex TFIID other proteins include TFIIB, TFIIA, TFIIF, TFIIE and TFIIH helicase activity in TFIIH unwinds DNA at the promoter kinase activity in TFIIH phosphorylates the polymerase at the CTD (carboxy-terminal domain), changing the conformation and enabling RNA pol II to start transcribing

protein factors required for initiation of translation in bacterial and eukaryotic cells

bacterial - IF-1, IF-2, and IF-3 eukaryotic - elF2, elF2B, elF3, elF4A, elF4B, elF4E, elF4G, elF5, and elF6

bacterial versus eukaryotic ribosomes

bacterial ribosome - 70S: 50S and 30S eukaryotic ribosome - 80S: 60S and 40S the fact that prokaryotic and eukaryotic ribosomes have slightly different structure is no small fact - this difference allows us to target antibiotics, like macrolides (azithromycin, erythromycin), tetracyclines (doxycycline), vancomycin, and others to bacterial cells with fewer side effects to humans drug targets - specifically attacking bacterial ribosome

structure of E. coli RNA polymerase holoenzyme

beta - main catalytic subunit; polymerase activity (helps growing of RNA strand) beta' - DNA binding alpha - assembly and binds to upstream promoter elements sigma - σ subunit binds initial DNA sequence omega - stabilizes polymerase from denaturation two identical alpha subunits

transcriptional activation domain

binding of RNA polymerase or other factors

DNA-binding domain

binds to a specific nucleotide sequence in the promoter region or to a DNA response element

elF4G

binds to elF4E and to poly(A) binding protein (PAB) part of the elF4F complex

elF4B

binds to mRNA facilitates scanning of mRNA to locate the first AUG

elF4E

binds to the 5' cap of mRNA part of the elF4F complex

ABO blood system

blood group - antigens on red blood cells; antibodies in the serum; can receive blood types 1. A - A; anti-B; A and O 2. B - B; anti-A; B and O 3. O - O; anti-A and anti-B; O 4. AB - A and B; none; A, B, and O

defects in synthetic enzymes of glycosaminoglycans

bone deformation - characteristic of multiple heredity exostoses disease resulting from a genetic inability to add the GlcNAc-GlcA disaccharide to the growing heparan sulfate or heparin chain - multiple large bone spurs develop

key concepts of transcription - transcription bubble

both strands of DNA can serve as template and different transcripts can be made from the same DNA sequence 3.6 x 10^4 bp DNA duplex unwinds, forming a "bubble" of ~17 bp 5' -> 3' replication

bacterial ribosome

bound tRNA's, rRNA, tRNA anticodons, protein - tRNA, mRNA, rRNA, proteins, E, P, and A positions 50S and 30S interface between small and large subunits - tRNA and mRNA puromycin site of peptide bond formation - mimics 3'-terminus of aminoacylated tRNA

naming monosaccharides

can be named on the basis of functional groups present: 1. ketone functional group - ketose 2. aldehyde functional group - aldose can be named on the basis of number of C-atoms in the main skeleton: 1. three carbons in the skeleton - triose 2. four carbons in the skeleton - tetrose 3. five carbons in the skeleton - pentose 4. six carbons in the skeleton - hexose ...so on and so forth combining the naming schemes: 1. triose + aldose -> aldotriose (glyceraldehyde) 2. hexose + aldose -> aldohexose (glucose) 3. hexose + ketose -> ketohexose (fructose)

peptidogylcans

carbohydrate + peptides - short sequences of amino acids, not proteins! sugar and amino acid polymers making up the cell wall of bacteria also known as murein - a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of most bacteria, forming the cell wall the sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) the peptide is a chain of three to five amino acids attached to the N-acetylmuramic acid - five amino acids = pentapeptide example - a peptidoglycan monomer consists of two joined amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), with a pentapeptide coming off of the NAM (L-alanine, D-glutamic acid, meso diaminopimetic acid, D-alanine, and D-alanine Cross-linking between amino acids in different linear amino sugar chains occurs with the help of the enzyme *transpeptidase! - transpeptidase is the target for several antibiotics like penicillins, cephalosporins etc. mesh-like structure is formed by cross-linking peptide chains for cell wall synthesis of bacteria by transpeptidase

carbohydrates - isomeric forms

carbohydrates exist in dazzling variety of isomeric forms - geometric or cis-trans stereoisomers optical isomerism - chiral carbons (number of stereoisomers = 2^n) all have the same molecular formula constitutional isomers - glyceraldehyde and dihydroxyacetone enantiomers - D-glyceraldehyde and L-glyceraldehyde most vertebrate monosaccharides have the D configuration! diastereomers - D-altrose and D-glucose epimers - D-glucose and D-mannose anomers - alpha-D-glucose and beta-D-glucose

messenger RNA (mRNA)

carries the information specifying the amino acid sequence of the protein to the ribosome transcribed from template DNA strands by RNA polymerase enzymes in the nucleus of cells the only type of RNA that contains information that is translated into protein it is read in three-nucleotide segments termed codons

sucrose catabolism

catabolism of sucrose starts with a hydrolytic cleavage catalyzed by the enzyme sucrase sucrose + sucrase -(H2O)> glucose + fructose both can enter glycolysis pathways cleaving the glycosidic bond - sucrase hydrolyzes bond

Guillain-Barré syndrome

caused by an autoimmune response to cell surface gangliosides, leading to acute quadriplegia - paralysis caused by illness or injury that results in the partial or total loss of use of all four limbs and torso treatment options - immunotherapy, pain medications, rehabilitation, etc.

pyruvate metabolism disorders

caused by the lack of ability to metabolize pyruvate

glycogen storage diseases

caused when there is a defect in the enzymes involved in metabolism of glycogen

hereditary fructose intolerance

causes due to a lack of the enzyme that is required to metabolize fructose

comparison between cellulose, starch, and glycogen

cellulose: 1. polysaccharide containing glucose monomer linked via β-1,4 linkages 2. β-configuration allows to form long straight chains 3. parallel chains interact with hydrogen bonding, forming fibrils, generating a rigid, supportive structure 4. cellulose cannot be used as a fuel source in most vertebrate animals as they lack enzymes to cleave the β-1,4 linkages starch and glycogen: 1. polysaccharides containing glucose monomer linked via α-1,4 linkages 2. α-configuration allows to form a hollow helix 3. the hollow helix is suited to the formation of compact accessible stores of sugar changing configuration = two completely different polysaccharides with different functions

the arrangement of the code is somewhat non-random

change in the 1st codon position tends to generate similar amino acids codons with pyrimidine at the 2nd position encode mostly hydrophobic amino acids those with purine encode mostly polar amino acids XYC, XYU often code for same amino acids XYG and XYA code the same amino acid with exception of two - UGA = stop and UGG = Trp UXY, CXY, AXY, GXY - similar, if not the same amino acid XCY or XUY - hydrophobic aa XGY or XAY - polar aa XYU or XYC - same aa XYG or XYA - same aa with the exception of two

essential components for step 5 of protein synthesis - folding and post translational processing

chaperones and folding enzymes - PPI, PDI specific enzymes cofactors other components for removal of initiating residues and signal sequences additional proteolytic processing modification of terminal residues attachment of acetyl, phosphoryl, methyl, carboxyl, carbohydrate, or prosthetic groups

fMet-tRNA(fMet)

charged tRNA with fMet linked aa + tRNA + ATP ➔ aminoacylated-tRNA + AMP + 2Pi ∆G'o = -29 kJ/mol

operon structure in prokaryotes

classic operons contain: 1. genes coding for the proteins of interest 2. operator site: - upstream of the genes - a nontranscribable region of DNA that is capable of binding a repressor protein 3. promoter site: - upstream of operator site - provides a place for RNA polymerase to bind DNA -> activator binding site -> promoter -> operator/repressor binding site -> genes A, B, C (genes to be transcribed as a unit) regulatory sequences - activator binding site and operator mRNA can be translated into 3 proteins - share one promoter E. coli - most gene expression is regulated by repression (inhibits RNA polymerase from moving forwards)

classifying carbohydrates

classified on the basis of number of sugar units present in these molecules monosaccharides, disaccharides, oligosaccarides, and polysaccharides

polyadenylation (polyA tail)

cleavage signal sequence indicates the position of cleavage RNA pol II synthesizes RNA beyond the cleavage signal sequence cleavage signal is bound by an endonuclease and a polyadenylate polymerase bound to carboxy terminal domain (CTD) endonuclease cleaves RNA 10−30 nt downstream to highly conserved AAUAA polyadenylate polymerase synthesizes 80−250 nt of polyA AAA(A)n-OH(3') = 80-250 adenosines

chondroitin 4- and 6-sulfates

composed of D-glucuronate (GlcA) and GalNAc-4- or 6-sulfate linkage is β(1,3) important component of cartilage

hyaluronates

composed of D-glucuronate (GlcA) plus N-acetylglucosamine (GlcNAc) linkage is β(1,3) component of connective tissue, synovial fluids, and the vitreous humor of the eye

heparin and heparan sulfates

composed of L-iduronate (IdoA: many with 2-sulfate) or D-glucuronate (GlcA: many with 2-sulfate) and N-sulfo-D-glucosamine-6-sulfate linkage is α(1,4) if IdoA, β(1,4) - L-iduronate-2-sulfate if GlcA - heparans have less overall sulfate than heparins; N-sulfo-GlcNAc-6-sulfate

homopolysaccharides

composed of a single type of sugar monomer (starch, glycogen) - act as storage forms of glucose (fuel) and cellulose (structural)

polysaccharides

contain many monosaccharide units in their structure examples - starch, cellulose, glycogen, etc. connected by glycosidic link/bond these are consumed in the diet, serve as energy reserves (glycogen in humans)

monosaccharides

contain one sugar molecule in their structure - the simplest carbohydrates are composed of C, H, and O examples - glucose, fructose (consumed in diet, primary energy)

oligosaccharides

contain short chain of monosaccharide (sugar) units in their structure - 3-20 sugars connected by glycosidic link/bond these are synthesized in the body

disaccharides

contain two monosaccharide (sugar) units in their structure examples - sucrose, lactose connected by glycosidic link/bond - O-glycosidic bond eliminates water (condensation reaction)

heteropolysaccharides

contain two or more different monosaccharide units (bacterial cell wall, hyaluronic acid) - found in neural tissues, connective, and epithelial tissues

PXR-mediated drug-drug interactions - example 1

contraceptives + Rifampicin -> unwanted pregnancy contraceptives are metabolized by CYP3A, which is induced by rifampicin - drug interaction that increases metabolism of contraceptives (less protection against pregnancy)

chemistry of transcription by RNA polymerase

stabilization of transition state as in DNA polymerases RNA pairs with DNA adding NTP with RNA - -OH group (3') attacks the NTP triphosphate group and forms a phosphoester bond (new NTP and growing strand) two Mg2+ ions help stabilize the transition state works similarly to DNA polymerase!

defects in degradative enzymes of glycosaminoglycans

defect in the degradative enzymes - results in accumulation of incompletely degraded glycosaminoglycans that produce diseases examples: 1. Scheie syndrome (moderate) - joint stiffening, but normal intelligence and life span 2. Hurler syndrome (severe) - enlarged internal organs, heart diseases, dwarfism, mental retardation and early death

fructokinase defect

defect in the gene encoding fructokinase causes fructosemia or fructosuria accumulation of fructose in the blood - excreted in urine no clinical symptoms, not nearly as fatal because no damage occurs to the liver cells

glucose 6-phosphatase defect

defect in the gene encoding glucose-6-phosphatase causes von Gierke's disease impairs the ability of the liver to produce free glucose from glycogen and from gluconeogenesis, causing severe hypoglycemia and results in increased glycogen storage in liver the primary treatment goal is prevention of hypoglycemia by frequent feedings of foods high in glucose or starch, which is readily digested to glucose

defects in glycosaminoglycans

defects in the synthesis or degradation of sulfated glycosaminoglycans can lead to serious human diseases the result can be any of a wide variety of defects in cell signaling, cell proliferation, tissue morphogenesis, or interactions with growth factors

galactose-1-phosphate uridyl transferase deficiency

deficiency causes galactosemia - galactose in blood galactosemia results in build up of galactose-1-phosphate and the disease manifests in newborns with acute liver failure - deadly if not diagnosed promptly treatment for classic galactosemia is eliminating lactose and galactose from the diet accumulating galactose in the blood - leads to acute liver failure, usually diagnosed in infancy

lactose intolerance

deficiency in lactase causes lactose intolerance people having lactose intolerance must avoid a diet containing milk and milk products alternatively, they can take the enzyme lactose in tablet form that is available buildup of lactose in the intestinal tract - leads to cramping and diarrhea

aldolase B defect

deficiency of the enzyme aldolase B causes hereditary fructose intolerance (HFI) HFT results in trapping of phosphate, resulting in reduced regeneration of ATP managed with careful dietary planning by avoiding foods that contain fructose fatal is buildup occurs in the liver

the genetic code is resistant to mutation

degenerate code allows certain mutations to still code for the same amino acid these are known as silent mutations - different nucleotide in DNA but same amino acid in protein mutation in the first base of a codon usually produces a conservative substitution - example: GUU → Val but AUU → Leu 64 combinations - 3 stop codons and 1 start codon

glycogenolysis

degradation of glycogen glycogen + glycogen phosphorylase -> glucose 1-phosphate + phosphoglucomutase -> glucose 6-phosphate + glucose 6-phosphatase -> glucose glycogen (n+1)-mer + glycogen phosphorylase -> alpha-D-glucose-1-phosphate + glycogen n-mer branched glycogen: glycogen + alpha-1,6-glycosidase (de-branching enzyme) -(H2O)> de-branched glycogen + glucose -> glycolysis or bloodstream glucose removed from the non-reducing end! glycogen phosphorylase cleaves the 1,4-glycosidic bonds glycosidase cleaves the 1,6-glycosidic bonds - debranching, malfunctioning or deficiency in the enzyme can occur

diabetes mellitus

diabetes mellitus refers to a group of diseases that affect how your body uses blood sugar (glucose) this is a chronic, metabolic disease characterized by elevated levels of blood glucose (or blood sugar), which leads, over time, to serious damage to the heart, blood vessels, eyes, kidneys, and nerves about 422 million people worldwide have diabetes, and 1.6 million deaths are directly attributed to diabetes each year in 2018, 34.2 million Americans had diabetes chronic diabetes conditions include type 1 diabetes and type 2 diabetes people with type 1 diabetes need insulin therapy to survive metformin (Glucophage, Glumetza, others) is generally the first medication prescribed for type 2 diabetes

epimers

differ at one of several asymmetric carbon atoms a type of diastereomer

constitutional isomers

differ in the order of attachment of atoms

orphan nuclear receptors (PXR, CAR) are regulators of drug metabolizing gene expression

drug A -> PXR + RXR -> CYP3A4 (liver, intestine) -> CYP3A enzyme drug B -(CYP3A enzyme)> HO-drug B drug A binds to PXR and activates expression of CYP3A - responsible for metabolism of many drugs drug A + drug B, which is metabolized by CYP3A - drug B is metabolized faster, not reaching the best therapeutic concentration (this is a drug-drug interaction) increase drug B dose when taking in combination prodrug - active metabolites; if metabolism is increased by drug A, active metabolite concentration increases in patient (possibility of toxicity or overdose)

Pregnane X receptor (PXR) and its functions

drug metabolism - target of PXR PXR: 1. phase I enzymes - metabolism 2. phase II enzymes - metabolism 3. transporters - absorption, distribution, and elimination metabolism - oxidation or conjugation -> O-glucuronide diverse drugs activate through heterodimer complex protect against xenobiotics - group of xenobiotic receptor with DNA-binding domain causes drug-drug interactions - influencing drug metabolism and PK in human body

DNA and RNA

each nucleotide contains a phosphate, a 5-carbon sugar molecule and a nitrogenous base

transcription elongation and termination

elongation factors bound to RNA pol II enhance processivity and coordinate post-translational modifications for termination, pol II is dephosphorylated regulation is complex

lacZ gene

encodes beta-galactosidase

lacY gene

encodes galactoside permease

lacA gene

encodes transacetylase

transcription in eukaryotes

eukaryotic cells have 3 types of RNA polymerases, each of which is responsible for synthesis of a special class of RNA different RNA polymerases recognize different sequences for initiation multiple proteins called transcription factors are involved in the coordination of RNA synthesis

trp operon

example of a repressible system regulator -> promoter -> operator -> structural regulator -> repressor - repressor cannot bind to operator by itself corepressor (end product, aka tryptophan) binds to repressor - repressor-corepressor complex binds to operator and represses enzyme synthesis high expression under normal conditions taking use of outside tryptophan, binding to repressor complex, and pausing gene expression original repressor is non-functional until turned on by the end product

gangliosides

glycolipid - carbohydrate + lipids (membrane lipid) are sialic acid containing glycosphingolipids are ubiquitously found in tissues and body fluids, and are more abundantly expressed in the nervous system the two hydrocarbon chains are embedded in the plasma membrane and the oligosaccharides located on the extracellular surface, are involved in cell-cell recognition, adhesion, cell signaling, etc. structure of GM1 ganglioside - common brain ganglioside; carbohydrate chain + ceramide (fatty acid + sphingolipid) sialic acid (Sia) - N-acetylneuraminate sphingosine backbone, amide bond fatty acid attachment, polar head group saccharide - sialic acid residues

four classes of introns

group I group II spliceosomal introns tRNA introns

lac operon

example of an inducible system RNA polymerase blocked from transcribing lac operon lacl mRNA - active form of repressor bound to operator lactose absent, repressor bound to operator, operon repressed - in the absence of lactose, the repressor remains bound to the operator, and RNA polymerase is therefore prevented from moving down the lac operon and transcribing the genes E. coli only likes glucose for energy - metabolizing lactose is toxic to E. coli genes responsible for transport, metabolism, and detoxifying of lactose metabolites - induced when in depletion of glucose, high in lactose to remove the repressor, an inducer (lactose metabolite) must bind the repressor protein so that RNA polymerase can move down the gene - lactose metabolite binds to and forms the inactive form of repressor as the concentration of the inducer increases, it will pull more copies of the repressor off from the operator region, freeing up those genes for transcription lactose present, repressor not bound to operator, operon depressed - in the presence of lactose, the repressor is converted to its inactive form, which does not bind to the operator (RNA polymerase can therefore move past the operator and transcribe the lacZ, lacY, and lacA genes into a single mRNA) positive feedback loops

alternative splicing in the calcitonin gene of rats

exon 1 -> intron -> exon 2 -> intron -> exon 3 -> intron -> exon 4 + poly(A) site -> intron -> CGRP -> exon 6 + poly(A) site cleavage and polyadenylation form two different products: 1. thyroid -(translation)> protease action -> calcitonin (calcium-regulating hormone) 2. brain -(translation)> protease action -> CGRP (calcitonin gene-related peptide) splicing forms two different mature mRNA products different proteins from the same gene in different rat tissues - cleaved at two different poly A sites

wobble hypothesis of Francis Crick

explanation for codon degeneracy transfer RNAs recognize codons by base pairing between the mRNA codon and anticodon the two RNAs are paired antiparallel 32 tRNAs -> 61 amino acid codons codons that differ in the 1st or 2nd base require different tRNAs

how do 2 guanines and 1 adenine = 1 glycine

forming proteins

lacl gene

forms lacl-mRNA -> repressor protein

Francis Crick's adaptor hypothesis

four types of nucleotides - A, G, C, U translation -> 21 different amino acids (eukaryotes) 1 nt - 1 aa; 4^1 = 4 2 nt - 1 aa; 4^2 = 16 3 nt - 1 aa; 4^3 = 64 4 nt - 1 aa; 4^4 = 256 61/64 codons code for amino acids - 3 (UAA, UGA, UAG) are stop codons one (AUG) is an initiation codon (as well as Met codon) the code is written in the 5' → 3' direction tRNA and mRNA - amino acid, amino acid binding site, adaptor and nucleotide triplet coding for an amino acid

fructose metabolism

fructose + fructokinase -(ATP -> ADP)> fructose-1-phosphate + aldolase B, aka fructose-1-P-aldolase) -> glyceraldehyde + DHAP (dihydroxy acetone phosphate) glyceraldehyde + triose kinase -(ATP -> ADP)> glyceraldehyde-3-phosphate glyceraldehyde-3-phosphate is an intermediate in glycolysis DHAP + triose-phosphate isomerase -> glyceraldehyde-3-phosphate 4 enzymes in reaction pathway - fructose -> glycolysis intermediates defects in these enzymes cause inadequate metabolism, leading to genetic diseases

glycoproteins

function in the structure, reproduction, immune system, hormones, and protection of cells and organisms mucins found in mucus - protect sensitive epithelial surfaces, including the respiratory, urinary, digestive, and reproductive tracts antibodies that bind to antigens in the immune responses proteins which contain oligosaccharide chains (glycans), covalently attached to amino acid side-chains occur in all forms of life and have functions that span the entire spectrum of protein activities - enzymes, transport proteins, hormones, structural proteins, etc. protein + carbohydrate - 60% protein, 40% carbohydrates

essential components for step 3 of protein synthesis - elongation

functional 70S ribosome - initiation complex aminoacyl-tRNAs specified by codons elongation factors - EF-Tu, EF-Ts, EF-G GTP Mg2+

retroviruses typically contain three genes plus a long terminal repeat

gag (group associated antigen) - encodes a long polypeptide that is cleaved into six smaller proteins that make up the viral core pol - encodes protease that cleaves the long polypeptide, reverse transcriptase, and an integrase to insert DNA into host genome env - encodes viral envelope long terminal repeat (LTR) facilitates integration of virus genome into host DNA

galactose metabolism

galactose + galactokinase -(ATP -> ADP)> galactose 1-P + galactose-1-phosphate uridyl transferase -(UDP-glucose -> UDP-galactose)> glucose 1-P + phosphoglucomutase -> glucose 6-P -> glycolysis UDP galactose -(UDP galactose epimerase)> UDP-glucose - responsible for the exchange of molecules (glucose for galactose), and the enzyme must be regenerated to UDP-glucose for further conversions first intermediate in glycolysis - G-6P

alpha-1,6-glycosidase defect

genetic defect in debranching enzyme leads to Cori's disease individuals cannot degrade glycogen completely and use their glycogen stores very inefficiently treatment involves a high-protein diet in order to facilitate gluconeogenesis accumulating glycogen in the liver - affects gluconeogenesis and causes an enlarged liver (gluconeogenesis is the production of glucose from non-carbohydrate starting material)

polysaccharides

glycogen, starch, cellulose, etc. are long chains of monosaccharides linked by glyosidic bonds two kinds - homopolysaccharides and heteropolysaccharides alpha-glucose structure - conformation

relationship between protein half-life and amino-terminal amino acid residue

stabilizing: 1. Met, Gly, Ala, Ser, Thr, Val - half-life >20 hours destabilizing: 1. Ile, Gln - ~30 minutes 2. Tyr, Glu - ~10 minutes 3. Pro - ~7 minutes 4. Leu, Phe, Asp, Lys - ~3 minutes 5. Arg - ~2 minutes

splicing mechanism

group III - spliceosome introns - > 50 proteins active spliceosome - small nuclear ribonucleoproteins; U1 and U2 snRNP and ATP then U2, U4/U6 + U5 and ATP forms the inactive spliceosome - lariat formation and intron release 5'-AU--//--AC-3' for U11/U12 spliceosomes on site splicing - coordinated transcription and splicing squeezes out adenine - acts as a nucleophile to attack the phosphodiester bond and expose hydroxy group to attach phosphodiester bond happens as transcription occurs

protein degradation

half-lives of proteins range from seconds to days to even months hemoglobin is long-lived defective proteins are short-lived, as are many metabolism regulatory proteins that respond to rapidly changing needs degradation for recycling

glycosaminoglycans

heteropolysaccharides - two different sugars -> disaccharide unit are historically referred to as the muco polysaccharides, given that they were originally characterized in mucus membranes and mucosal exudates are long, unbranched polysaccharides containing a repeating disaccharide unit the disaccharide units contain either of two modified sugars, N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc), and a uronic acid, such as glucuronate (GlcA) or iduronate (IdoA) bind to a variety of extracellular ligands, thereby modulating the ligands interactions with specific receptors on the cell surface proteins bound to glycosaminoglycans are called proteoglycans! components of connective tissue, cartilage, arterial walls, etc.

galactosemia

high level of galactose in the blood caused by lack of an enzyme necessary for metabolizing galactose

HDAC inhibitors

histone acetylation induced by histone acetyl transferases (HATs) is associated with increased gene transcription histone hypoacetylation induced by histone deacetylase (HDAC) activity is associated with gene silencing HDAC inhibitors affect histone acetylation - makes transcription more active HDAC inhibitors block growth of tumors by inducing cell cycle arrest, (terminal) differentiation of tumor and/or apoptosis Vorinostat (SAHA), Mocetinostat (MGCD0103), and Abexinostat (PCI-24781) - -C(=O)NH-OH interacts with Zn2+ in HDAC's catalytic site zinc catalysis of deacetylation

histone acetylation

histone acetyltransferases (HATs) acetylate a particular Lys in histone protein acetylation of Lys results in decreased affinity of histone for DNA - decreases the positive charge on lysine residues chromatin remodeling engine binds to acetylated Lysol residues and reconfigures the nucleosome to expose sites for additional transcription factors reversed by histone deacetylases (HDACs) that make chromatin inactive - DRUG TARGET detaching DNA from histone protein to be open for transcription - histone regain positive charge to suppress gene expression by HDACs (treatment of tumors)

erythropoetin (EPO)

hormone that is a glycoprotein - glycoprotein present in the blood serum secreted by kidneys and stimulates the production of red blood cells composed of 165 amino acids and is N-glycosylated at three asparagine residues and O-glycosylated on a serine residue mature EPO is 40% carbohydrate by weight and glycosylation enhances stability of protein in blood

steps that regulate steady-state concentration of protein

how much primary RNA transcript to make how to process this RNA into mRNA how rapidly to degrade the mRNA how much protein to make from this mRNA how efficiently to target the protein to its location how to alter the intrinsic activity of this protein how rapidly to degrade the protein DNA -(transcription initiation)> primary RNA transcript -(posttranslational processing)> mRNA and RNA stability -(translational regulation)> protein -> protein modification -> modified protein -> protein transport -> protein degradation

vesicle-based translocation into ER

human influenza virus A, human preproinsulin, bovine growth hormone, bee promellitin, and Drosophila glue protein all contain basic aa, hydrophobic core, and cleavage site directing eukaryotic proteins with the appropriate signals to the ER the signal sequence includes a hydrophobic core, which is preceded by one or more basic residues the polar and short-side-chain residues immediately preceding to the left of the cleavage sites highly conserved - entering memory system of the cell

uses of recombinant EPO

human recombinant EPO is being used to treat anemia -particularly induced by cancer chemotherapy in chemotherapy, RBC count falls very low (anemia) athletes use recombinant EPO to increase red blood cell count and hence oxygen carrying capacity for better delivery of oxygen to the muscle tissue, boosting athletic performance - prohibited EPOs (disqualified if used)

open reading frame (ORF)

in a random sequence of nucleotides, one in every 20 codons in each reading frame, on average, will be a termination codon (3/64) where a reading frame exists without a termination codon for 50 or more codons, the region is called an open reading frame long open reading frames usually correspond to genes that encode proteins an uninterrupted gene coding for a typical protein with a molecular weight of 60,000 would require an open reading frame with 500 or more codons

HIV genome

in addition to the typical retroviral genes, HIV contains several small genes with a variety of functions some of these genes overlap alternative splicing mechanisms produce many different proteins from this small (9.7 x 103 nucleotides) genome

key concepts of transcription - RNA polymerase

it is not critical for RNA polymerases to be as accurate as DNA polymerases as the DNA is rewound, the RNA-DNA hybrid (~ 8 bp) is displaced and the RNA strand is extruded RNA polymerase lacks 3' -> 5' exonuclease, so error rate is high - 10^-4 to 10^-5 per base added cannot proofread RNA strand - not as many consequences when errors occur (degraded in the cell, much faster reaction) gene - several thousand BP's of RNA that get translated into proteins

summary of RNA processing

in eukaryotes, messenger RNAs are formed from primary RNA transcripts - primary RNA transcripts often contain noncoding regions called introns, which are removed by splicing group I introns are found in rRNAs and their excision requires a guanosine cofactor - group I and some group II introns are capable of self-splicing; no protein enzymes are required nuclear mRNA precursors have a third class of introns that are spliced with the aid of RNA-protein complexes called snRNPs the fourth class of introns, found in some tRNAs, are the only ones known to be spliced by protein enzymes messenger RNAs are also modified by addition of a 7-methylguanosine residue at the 5' end, and cleavage and polyadenylation at the 3' end to form a long poly(A) tail ribosomal RNAs and transfer RNAs are made from longer precursor RNAs that are trimmed by nucleases, and some bases are modified enzymatically to yield the mature RNAs spliceosome involved in RNA processing

biomolecules of eukaryotic protein synthesis

in eukaryotes, protein synthesis requires >300 biomolecules: - >70 ribosomal proteins - ~20 amino acid activation enzymes - ~20 protein factors for initiation, elongation, and termination of peptides - ~100 additional enzymes for final processing of proteins - ~40 kinds of tRNAs and rRNAs

control of gene expression in eukaryotes

in eukaryotic cells, positive regulation predominates in regulation of gene expression transcription Factors enhancers regulation of chromatin structure - histone acetylation and DNA methylation controlling what genes are expressed in different cells

summary of stage 3 - elongation

in the subsequent elongation steps, GTP and elongation factors are required for binding the incoming aminoacyl-tRNA to the aminoacyl site on the ribosome in the first peptidyl transfer reaction, the Met residue is transferred to the amino group of the incoming aminoacyl-tRNA movement of the ribosome along the mRNA then translocates the dipeptidyl-tRNA from the aminoacyl (A) site to the peptidyl (P) site, a process requiring hydrolysis of GTP APE sites

extension of central dogma

includes RNA-dependent synthesis of RNA and DNA DNA -> DNA by DNA replication RNA -> DNA by reverse transcription DNA -> RNA by transcription RNA -> RNA by RNA replication RNA -> protein by translation RNA product of genes - secondary messenger using RNA as genomic material to synthesize DNA and RNA - more complicated central dogma

influenza

influenza A viruses recognize sialic acid residues of gangliosides and glycoproteins on cell surfaces as receptor molecules for invasion of host cells influenza viruses bind through hemagglutinin onto sialic acid sugars on the surfaces of epithelial cells, typically in the nose, throat, and lungs of mammals, and intestines of birds an influenza virus infects a host cell when hemagglutinin grips onto glycans on the cell surface Umifenovir (arbidol) treatment for influenza infection used in Russia and China - inhibits membrane fusion perfect example of cell-cell recognition - treatment not approved in US, but prevents infection of the influenza virus

eukaryotic translation initiation

initiation factor - nine (eIF4F complex - eIF4E, eIF4G, eIF4A) a lot of initiation factors ribosome - 40S and 60s (different ribosomal subunits) initiation sequence at 5' end - 5' cap poly A tail at 3' end - present chain initiating amino acid - methionine placement of AUG codon P-site - scanning of mRNA (find AUG and bringing in large subunit)

prokaryotic translation initiation

initiation factor - three (IF3, IF2, IF1) ribosome - 30S and 50S initiation sequence at 5' end - Shine-dalgarno sequence (no 5' cap) poly A tail at 3' end - absent chain initiating amino acid - N-formyl methionine placement of AUG codon P-site - by Shine-dalgarno sequence (AUG places away from Shine-dalgarno sequence)

summary of initiation in eukaryotes

initiation of protein synthesis involves formation of a complex between: 1. the 40S ribosomal subunit 2. mRNA 3. GTP 4. Met-tRNA 5. initiation factors 6. the 60S ribosomal subunit GTP is hydrolyzed to GDP and Pi uses more initiation factors than E. coli - over 12, including eIFIA and eIF3 (functional homologs of IF-1 and IF-3) require 5′ cap structure and has a step that circularizes the mRNA

insertion mutation

inserting an unwanted amino acid into the sequence of mRNA alters the reading frame

splicing

introns are found in most genes most genes in vertebrates, some in yeast, a few bacteria have introns (non-coding information) axons usually < 1000 bp in length introns 50−20,000 bp in length some genes have dozens of introns specific and precise 10% of sequence are exons in DNA

diastereomers

isomers that are not mirror images epimers and anomers

anomers

isomers that differ at a new asymmetric carbon atom formed on ring closure a type of diastereomer cyclized forms of linear glucose introduce chirality - alpha and beta conformations alpha - anomeric carbon substitution is below the plane of the ring beta - the substitution is above the plane of the ring

peptidoglycan function

peptidoglycans serve a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm peptidoglycan is also involved in binary fission during bacterial cell reproduction

bacterial operon

lacI, lacZ, lacY and lacA when glucose is abundant and lactose is lacking, cells make only very low levels of enzymes for lactose metabolism lactose enters the cell through galactoside permease inside the cell, lactose triggers beta-galactosidase: 1. isomerization (minor pathway) -> allolactose 2. hydrolysis (major pathway) -> galactose + glucose DNA -> PI -> lacl gene -> Plac (promoter) -> operator -> lacZ gene -> lacY gene -> lacA gene regulatory gene includes PI and lacl gene, and lac operon includes everything from promoter on under normal conditions - low levels

glycogen phosphorylase defect

lack in muscle cells causes McArdle's disease individuals have little tolerance for physical exercise supervised exercise programs have been shown in small studies to improve exercise capacity oral sucrose treatment taken 30 minutes prior to exercise has been shown to help improve exercise tolerance

lactose catabolism

lactose is metabolized by breaking it down into galactose and glucose by the enzyme lactase lactose (galactose + glucose) + lactase -> galactose + glucose both can enter glycolysis pathways lactase cleaves the glycosidic bond

regulated gene

levels of the gene product rise and fall with the needs of the organism such genes are inducible - able to be turned on and repressible - able to be turned off in response to stimuli - very dynamic

determining reducing end of a carbohydrate

look at the anomeric carbon and the -OH group attached if it is free, it can open up and convert back to the linear structure and act as a reducing agent

protein degradation - 3 major mechanisms of degradation

lysosomal protein degradation (proteolysis) - intracellular digestion of proteins by proteases apoptosis programmed cell death mediated by caspases - cysteine aspartyl proteases ATP-dependent proteolysis in proteasome - ubiquitin-dependent protein degradation

RNA processing is RNA species dependent

mRNA (messenger RNA): 1. 5' capping 2. 3' polyadenylation 3. splicing - intron removal 4. alternative splicing rRNA (ribosomal RNA) 1. splicing - self-splicing tRNA (transfer RNA): 1. removal of the extra nucleotides 2. 3' addition of CCA - responsible for the transfer of amino acids and modifying nucleotides 3. nucleotide modification each type of rna gets processed differently to become mature rna

essential components for step 2 of protein synthesis - initiation

mRNA - template N-formylmethionyl-tRNA(fMet) initiation codon in mRNA - AUG 30S ribosomal subunit 50S ribosomal subunit initiation factors - IF-1, IF-2, IF-3 GTP Mg2+

the triplet, non-overlapping code

mRNA -> protein can occur during DNA replication - mutations insertion and deletion mutations - mutations that affect the reading frame when insertion and deletion occur simultaneously - reading frame may be restored, but not the right peptide (2 amino acids will be different) 3 amino acid codon - no overlapping of genetic code frame shift mutations requires inserting or deleting of a nucleotide that alters the reading frame of codons - loss of function mutations

RNA polymerase III

makes tRNAs and some small RNA products

transfer RNA (tRNA)

match specific amino acids to triplet codons in mRNA during protein synthesis responsible for converting the language of nucleic acids to the language of amino acids and peptides - free -OH links to peptide to synthesize each tRNA molecule contains a folded strand of RNA that includes a three-nucleotide anticodon the anticodon recognizes and pairs with the appropriate codon on an mRNA molecule while in the ribosome 40-60 tRNAs - acceptor stem, TC loop, anticodon loop (de-coder), D loop coding system: 1. converts nucleic acid language to peptide language 2. bringing amino acid to the right position - only mRNA to proteins

alternative splicing, cleavage, and polyadenylation

maximizing the use of coding sequence alternative splicing with multiple splicing sites - 5' splice site, 3' splice site, and poly(A) site forms mature mRNA poly(A) site choice - A1 or A2; primary transcript, then cleavage and polyadenylation at A1 or A2 forms different mature mRNA

covalent modification of histones

methylation phosphorylation acetylation ubiquitination sumoylation - similar to ubiquitination occur mostly in the N-terminal domain of the histones found near the exterior of the nucleosome particle nucleosome - histone octomer + 147 bp long DNA strand (binding due to difference in charge)

mRNA processing

modulate splicing, inhibit translation, using endogenous miRNAs or compete to bring up gene expression

positive regulation

molecular signal causes binding of activator to DNA, inducing transcription RNA polymerase II must be activated to translate genes

mRNA in eukaryotes

monocistronic made in the nucleus primary transcript must be processed to became mRNA

splice-switching oligonucleotides (SSOs) modulate alternative splicing

splicing factor A and splicing factor B inhibition - exon skipping (1-3) stimulation - exon inclusion (1-2-3)

common monosaccharides have cyclic structures

monosaccharides in solution exist in cyclic forms - these open sugars cyclize into rings in solution D-glucose (open chain form) <-> alpha or beta D-glucose (ring form) - intramolecular hemiacetal formation in glucose where aldehyde group is masked as a hemiacetal (H connected to anomeric carbon) D-fructose <-> alpha or beta D-fructose - intramolecular hemiketal formation in fructose where the keto group is masked a hemiketal (CH2OH connected to anomeric carbon) in solution, the cyclic (Haworth) and open structures (fisher projection) are in equilibrium! aqueous in solution - high percentage of water

heparin sulfate bridges in the anticoagulant pathway

negatively charged sulfates and carboxylates of heparin sulfate bind to positively charged Arg and Lys resides in the protein - binds to the residues and bridges proteins to interact and inhibit coagulation consequently, the affinity of antithrombin for thrombin is three orders of magnitude greater in the presence of heparin sulfate

enantiomers

non superimposable mirror images

overlapping vs. non-overlapping genetic code

non-overlapping code has only one frame to reach codons overlapping code - different transcription starting position could cause potential overlapping codons the genetic code used in all living systems is now known to be non-overlapping implies a specific "reading frame" - transcription can start or stop at different codons but generally not at different sites within a codon

formation of sucrose

non-reducing disaccharide sugar - table sugar; does not have a free hydroxy group on the anomeric carbon stable to oxidation - storage and transport of energy in plants alpha-D-glucose + beta-D-fructose -> sucrose + H2O condensation reaction - -OH reacts to form water from two monosaccharides connected by alpha-beta-1,2-glycosidic bond disaccharide formed from the reaction between α-D-Glucose and β-D-Fructose

30S subunit of ribosome

number of different proteins - 21 total number of proteins - 21 protein designations - S1-S21 number and type of rRNAs - 1 (16S rRNA)

50S subunit of ribosome

number of different proteins - 33 total number of proteins - 36 protein designations - L1-L36 (do not correspond to 36 different proteins) number and type of rRNAs - 2 (5S and 23S rRNAs)

TFIIH

number of subunits - 12 subunits Mr - 35,000-89,000 functions - unwinds DNA at promoter - helicase activity; phosphorylates polymerase II within the CTD; recruits nucleotide-excision repair proteins checks mutations

dietary carbohydrates and diseases

obesity, diabetes, cardiovascular diseases, dental cavities, etc. type II diabetes - insulin resistance by high glycemic carbohydrates disorders in carbohydrate metabolism lead to diseases

heparin

occurs in the intracellular granules of mast cells that occur in arterial walls heparin is sulfonated - anticoagulant properties (drugs) binds tightly to spike protein of coronavirus (used to infect healthy cells)

cell recognition - role of carbohydrates in the cell

one well-known example of cell-cell recognition mediated by carbohydrates is the ABO blood type system carbohydrate antigens (oligosaccharides) present on the surface of the red blood cells determine a person's blood type if an antigen that is not present in a person is introduced, the immune system recognizes it as foreign - red blood cells lyse rapidly, leading to severe drop in blood pressure, shock, kidney failure, and death from circulatory collapse different blood types are present in human population to prevent parasitic mimicry and corresponding selective pressure on parasite to enhance mimicry - driving evolution of diversity of surface antigens within human population!

potential RNA-targeted druggable genome

only ~1.5% of the genome encodes proteins - corresponding to ~20,000 proteins an estimated 10-15% of proteins are thought to be disease-related - ~2,000-3,000 proteins; encoded by 0.2% of the genome currently approved drugs interact therapeutically with <700 of these proteins - encoded by 0.05% of the human genome targeting RNAs could expand on the proportion of the human genome that could be therapeutically targeted physiological drug - high dose without severe toxicity using drug to modulate gene expression 30,000 protein coding genes - over 200,000 proteins majority of targets are not able to be modulated by chemical compounds (small molecules) - solve delivery problem of oligonucleotide drugs

control of gene expression in prokaryotes

operon structure inducible systems repressible systems

overview of mRNA processing

ovalbumin gene -> L - targets ovalbumin for cell export -> transcription and 5' capping -> splicing, cleavage, and polyadenylation + extra RNA -> mature mRNA

sources of carbohydrates in diet

plants produce carbohydrates! - primary source of carbohydrates are plant derived products by a process called photosynthesis photosynthesis - O2 and carbohydrates essential for survival (liberate molecules from environment) 6CO2 + 6H2O + energy -> C6H12O6 + 6O2 (simplest sugar) when consumed by living organisms, they are oxidized in the living cells to produce CO2, H2O, and energy C6H12O6 + 6O2 -> 6CO2 + 6H2O + energy - CO2 and H2O eliminated back to surroundings

ribonucleic acids

play several less-well understood functions in eukaryotic cells also act as genomic material in viruses other types of RNA include microRNA and long non-coding RNA

mRNA in prokaryotes

polycistronic made in the cytoplasm primary transcript is mRNA

summary of stage 5 - post translational modification

polypeptides undergo folding into their active, three-dimensional forms many proteins also are further processed by post translational modification reactions

inducible system

positive feedback allows gene products to be produced only when they are needed in inducible systems, the repressor is bound tightly to the operator system and thereby acts as a roadblock RNA polymerase is unable to get from the promoter to the structural gene because the repressor is in the way inducible systems are sometimes referred to as positive control mechanism

foot printing to find a DNA promoter binding site

premise - DNA bound by protein will be protected from chemical cleavage at its binding site 1. isolate a DNA fragment thought to contain a binding site 2. radiolabel the DNA 3. bind protein to DNA in one tube - keep another as a "naked DNA" control 4. treat both samples with chemical or enzymatic agent to cleave the DNA 5. separate the fragments by gel electrophoresis and visualize bands on X- ray film or imager plate defining new genes - finding the promoter region using RNA polymerase in E. coli by treating with DNase to cut the genome RNA polymerase protects DNA from being cut

ricin

present in castor plant seed targets and catalyzes depurination of A4324 that is contained in a highly conserved sequence of 12 nucleotides universally found in eukaryotic ribosomes a single activated ricin molecule in the cytosol is capable of depurinating ~1500 ribosomes/min used in the umbrella murder 22 μg/kg - 1.78 mg adult

eukaryotic transcription

promoter - many different promoter elements: TATA box, initiator elements, downstream core promoter element, CAAT box, and the GC box, etc. more complicated promoters in eukaryotes - one mRNA -> one gene RNA polymerase - three types of RNA polymerases, I, II, and III transcription initiation - form an initiation a complex with the various transcription factors that dissociate after initiation is completed; RNA is first transcribed in the nucleus and then translated in the cytoplasm post-transcriptional modifications - undergo post-transcriptional modifications including: capping, polyadenylation, and splicing monocystronic versus polyscystronic - eukaryotes contain mRNAs that are monocystronic transcription termination - transcription is terminated by two elements: a poly(A) signal and a downstream terminator sequence

prokaryotic transcription

promoter - three different promoter elements: -10, -35 promoters, and upstream elements one mRNA -> lots of genes RNA polymerase - one type of RNA polymerases transcription initiation - no such structure; transcription and translation occurs simultaneously in prokaryotes post-transcriptional modifications - do not occur in prokaryotes monocystronic versus polyscystronic - mRNAs in prokaryotes tend to contain many different genes on a single mRNA, meaning they are polycystronic transcription termination - termination is done by either rho-dependent or rho-independent mechanisms

eukaryotic initiation of protein synthesis

protein complex in the formation of a eukaryotic initiation complex involves circularization of mRNA UTR - 3' untranslated region PAB - poly(A) binding protein 5' cap, poly A tail, 40S, elF3, elF4F (elF4E, elF4G, and elF4A), and AUG

protein synthesis is highly energy-demanding, so it is highly coordinated

protein synthesis can use 90% of the chemical energy of a cell number of copies of protein produced corresponds to number of copies needed proteins are targeted to specific cellular locations degradation keeps pace with synthesis (steady-state) unless protein synthesis is induced or suppressed

second stage of protein synthesis - initiation of translation

proteins are made at the membrane of endoplasmic reticulum in the cytoplasm of prokaryotes and eukaryotes lumen - area enclosed by the ER membrane rough ER

summary of protein targeting and degradation

proteins are sorted based on their structure and delivered to their destination for secretory proteins, their signal is recognized by the signal recognition particles (SRP) - vesicle based proteins targeted to nucleus have an internal signal sequences that, unlike other signals, are not cleaved once the protein is in the nucleus - non-vesicle based all proteins are eventually degraded, using specialized proteolytic systems such as proteasome the amino acid residues on N-terminal of a protein determine the half life of the protein

direction of protein synthesis

proteins are synthesized from the N- to C-terminus - Howard Dintzis Experiment 3H-Leucine + cells (reticulocytes) t = 4, 7, 60 min -> purification of the completed alpha chain of hemoglobin dark red is radioactive sequence at early timepoints radioactivity is incorporated fully only into proteins that are almost fully synthesized

endocytosis (protein importation) pathways in eukaryotic cells

proteins can bind to receptors in coated pits Dynamin - pinches off vesicle from membrane fusion with endosomal membrane - caveosome coated pit on the cytosolic face of plasma membrane Clathrin-dependent endocytosis, Caveolin-dependent endocytosis, Clathrin- and caveolin-independent pathways

protein targeting

proteins move from site of synthesis to: 1. exit a cell 2. become part of the membrane 3. enter a subcellular compartment, etc. most have a signal sequence at or near N-terminus signal sequence is often removed when protein reaches its destination two different systems - vesicle and non-vesicle based

HbA1c test

reaction of glucose with hemoglobin - glucose binds to hemoglobin and remains in circulation for an average of 3-4 months transporters in the erythrocyte membrane equilibrate intracellular and plasma glucose concentrations so Hb is constantly exposed to glucose at whatever concentration is present in blood the primary amine group in Hb reacts with glucose to form the Schiff base - Schiffs base undergoes rearrangement to form the ketoamine ketoamine cyclizes to form glycated hemoglobin(GHB) - GHB remains in circulation, and the amount of modified Hb corresponds to the long term regulation (over several months (~3months) of glucose levels) in nondiabetic people, less than 6% of Hb is glycosylated; whereas, in uncontrolled diabetics, almost 10% of the Hb is glycosylated - 6-6.4% = prediabetes and >6.4% = diabetes sugars could also react with the N-terminus of other proteins to form advanced glycosylation products - AEGs that could alter protein function! AEGs implicated in aging, arteriosclerosis, damage to kidneys, etc. role of the protein hemoglobin is not compromised when glycated - if left untreated, damage to the nerves and eyes can occur

formation of lactose

reducing disaccharide sugar - found in milk and milk-containing products; has a free hydroxy group on the anomeric carbon (can open up ring) beta-D-galactose + alpha-D-glucose -> alpha-lactose connected by beta-1,4-glycosidic bond disaccharide formed from the reaction between one molecule of β-D-Galactose and one of either α or β-D-Glucose

formation of maltose

reducing disaccharide sugar - has a free hydroxy group on the anomeric carbon (can open up ring) alpha-D-glucose + alpha-D-glucose -> alpha-maltose connected by alpha-1,4-glycosidic bond formed by reaction between two of the same monosaccharides

some post-translationally modified amino acids associated with folding, processing, and targeting

removal of additional amino-terminal and carboxyl-terminal residues (the N- formylmethionine or methionine) acetylation of the amino-terminal residues modification of carboxyl terminal residues loss of signal sequences modification of individual AAs

RNA polymerase II

responsible for synthesis of mRNA and lncRNA very fast - 500-1000 nucleotides/sec specifically inhibited by mushroom toxin α-amanitin can recognize thousands of promoters binds to the initiation site and starts replication: 1. assembly of transcriptional factors IIH play a functional role of phosphorylating the carboxyl terminal (CTD) of polymerase II 2. factor F creates transcription bubble 3. elongation factors - 5' caps 4. terminational factors de-phosphorylates polymerase II and finds a new DNA to start the cycle over again

retroviruses can make DNA from RNA

retroviruses have genomes of ssRNA and the enzyme reverse transcriptase virus enters host cell - reverse transcriptase makes ss-DNA from the RNA then degrades the RNA strand from the DNA-RNA hybrid and makes a complementary DNA strand ds-DNA can then be incorporated into host DNA from RNA to DNA lifetime diseases - HIV

pharmaceutical targets for HIV - antiretroviral drugs

reverse transcriptase inhibitors: 1. nucleotide or nucleoside analog 2. drug names tend to end in "dine"or "sine" - Zidovudine (AZT), Didanosine (Videx), etc. protease inhibitors: 1. since proteases used in cleaving polyproteins for packaging into new viral particles 2. drug names tend to end in "avir" - Indinavir, Saquinavir, etc. enzyme specific to virus - higher affinity for reverse transcriptase, leave our own DNA and RNA polymerase alone

non-vesicle based protein targeting

ribosomes -> cytosol (retention) -> mitochondria, nucleus, or peroxisomes proteins for nucleus have a nuclear localization sequence (NLS) the NLS is not cleaved after the protein is targeted NLS binds importin alpha and beta and a GTPase called Ran complex docks at a nuclear pore and is imported no ribosomes in nucleus - NLS amino acid sequence to bring the protein in through nuclear pores

vesicle-based protein targeting

ribosomes -> endoplasmic reticulum (retention) -> golgi (retention) -> lysosomes, cell surface, or secretory vesicles as the peptide emerges from the ribosome, signal sequence is bound by signal recognition particle (SRP) SRP/ribosome/peptide complex delivered to the ER lumen - some modification takes place here (glycosylation, etc.) transport vesicles then take proteins to Golgi apparatus - proteins are further modified and sorted in Golgi guiding the N-terminal to the ER lumen

glycolipids

role is to maintain the stability of the cell membrane and to facilitate cellular recognition, which is crucial to the immune response and in the connections that allow cells to connect to one another to form tissues glycosphingolipids - located in nervous tissue and are responsible for cell signaling biomolecules containing one or more carbohydrate residues linked to a hydrophobic lipid moiety through a glycosidic linkage

group I and group II introns

self-splicing require no additional proteins or ATP in nuclear, mitochondrial, and chloroplast genomes differ mainly in the splicing mechanism

common features of transcription in prokaryotes and eukaryotes

sense (coding) strand antisense (noncoding) strand regulatory sequences - listed by the coding strand sequence promoter regions - RNA polymerase binds here on the coding strand

vitamin K

serves as an essential cofactor for a carboxylase and coagulation fat soluble compound cofactor for the carboxylation of glutamic acid residues a deficiency in Vitamin K results in impaired blood clotting and possibly bleeding the anticoagulant Warfarin inhibits Vitamin K too much vitamin K leads to a clotting risk - taking warfarin to decrease the potency of vitamin K in the body

microRNAs function in gene regulation

short noncoding RNAs of ~22 nucleotides bind to specific regions of mRNA to alter translation of mRNA assist in cleaving the mRNAs or block the mRNA from being translated ~1% of the human genome may encode miRNA synthesized from larger precursors - processed by two endoribonucleases, Drosha and Dicer

oligosaccharides

short polymers of several monosaccharides (less than 20), usually linked to a lipid or protein by N or O glycosidic bonds (glycoconjugates) N-linked oligosaccharide attached to asparagine - N atom connecting protein and sugar O-linked oligosaccharide attached to serine/threonine - O atom connecting protein and sugar chemical structure of glycolipids - glycolipids, glycero-glycolipids, and sphingo-glycolipids (glycolipids form the ABO blood groups) usually 3-20 monosaccharides long

different type of mutations

silent mutation - codes for same amino acid; mutation in the third position of the codon is usually silent missense mutation - codes for a different aa; mutation in the first or second position of the codon is usually missense nonsense mutation - codes for a stop codon, early termination of protein (mutation generating a stop codon) frameshift mutation (1bp deletion) - results in different aa for the rest of the protein, may result in early termination (insertion of deletion of a nucleotide)

regulation of RNA splicing

similar to DNA transcription, RNA splicing is regulated by splicing factors and corresponding cis-acting regulatory sequence on the pre-mRNA trans-acting splicing factors including *slicing repressors and *slicing activators - similar to transcription factors cis-acting regulatory sites - similar to promoter: 1. splicing silencers are sites to which splicing repressor proteins bind, reducing the probability that a nearby site will be used as a splice junction 2. splicing enhancers are sites to which splicing activator proteins bind, increasing the probability that a nearby site will be used as a splice junction RNA splicing for disease treatment

blood glucose measurement

single time measurements do not reflect the average blood glucose over hours and days, so dangerous increases may go unnoticed the average glucose concentration can be assessed by looking at its effects on hemoglobin - the oxygen carrying protein, by using the HbA1C test

conservation of 2 structure in small subunit rRNAs from bacteria, archaea, and eukaryotes and structure of bacterial and eukaryotic ribosome

small subunit bacteria - 50S and 30S; RNA, protein, cleft, and RNA yeast - 60S and 40S; RNA, protein, cleft, and RNA majority of ribosomal subunits have been conserved between bacteria, archaea, and eukaryotes - very small divergences the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure - evolution of change occurred without major structural differences

footprinting RNA polymerase on DNA

solution of identical DNA fragments radioactively labeled at one end of one strand - DNase I treat with DNase under conditions in which each strand is cut once (on average) - no cuts are made in the area where RNA polymerase has bound isolate labeled DNA fragments and denature - only labeled strands are detected in the next step separate fragments by polyacrylamide gel electrophoresis and visualize radio labeled bands on x-ray films uncut DNA fragment - missing bands indicate where RNA polymerase was bound to DNA partial DNase I digestion - ~10 TATAAT and 35 TTGACA regions bound by RNA polymerase

retrovirus oncogene

some retroviruses contain an oncogene - allows uncontrolled growth; cancer example - Rous sarcoma virus has the src gene: 1. Src for sarcoma, a cancer of bone, fat, muscle, etc. (vs. cancer of epithelial cell origin) 2. encodes a non-receptor tyrosine kinase, an enzyme that affects cell division

transcription inhibitor drugs

specifically target prokaryotic transcription - does not affect eukaryotes (antibiotics) rifampicin inhibits RNA synthesis by binding to the β subunit (main catalytic site) of bacterial RNA polymerases, preventing the promoter clearance step of transcription multi-drug resistant (MDR) TB - resistant to the 2 most powerful first line anti-TB drugs actinomycin D and acridine inhibit transcription by intercalating into the dsDNA between successive G-C bp's, preventing movement of the polymerase along the template - incorporates into DNA (not selective, can affect eukaryotes) α-amanitin (death cap mushroom) disrupts mRNA formation in mammalian cells by blocking pol II and pol III by interferring with the translocation of RNA and DNA needed to empty the site for the next round of RNA synthesis

exploit the RNA splicing to treat spinal muscular atrophy (SMA)

spinal muscular atrophy (SMA) is a rare neuromuscular disorder characterised by loss of motor neurons and progressive muscle wasting, often leading to early death bulbar and brainstem involvement - weak cry, tongue fasciculation breathing difficulties cardiac autonomic involvement and congenital heart defects gastroesophageal reflux constipation, weight loss joint contractures decreased or absent deep reflexes muscular atrophy, weakness and hypotonia

antisense based drug change SMN2 alterative splicing to treat SMA

spinraza (nusinersen) spinraza list price is $125,000 per injection, which puts the treatment cost at $750,000 in the first year and US $375,000 annually after that according to the New York Times, this places Spinraza "among the most expensive drugs in the world" including exon 7 in mRNA - increasing survival from disease 4-5 injections in the first year not a permanent fix - must take for lifetime or go back to diseased condition

spliceosomal introns

spliced by enormous complexes called splicesomes the most common introns frequent in the midst of protein-coding regions of eukaryotic genomes

tRNA introns

spliced by protein-based enzymes primary transcript cleaved by endonuclease exons are joined by ATP-dependent ligase highly ATP driven process

spliceosome

spliceosome introns are removed via a large complex called a spliceosome spliceosome made up of snRNPs - "snurps" for small nuclear ribonuclear proteins 5 snRNAs known in eukaryotes - U1, U2, U4, U5, U6 U6 - loading control of nuclear RNA in experiments GU at 5' end and AU at 3' end in mRNA usually mark sites of splicing

stage 3 - elongation

step 1 - initiation complex + incoming aminoacyl-tRNA -> binding of incoming aminoacyl-tRNA step 2 - peptide bond formation of amino acid connected to tRNA in A site with existing amino acid in P site -> deacylated tRNA(fMet) + dipeptidyl-tRNA2 in P site step 3 - translocation of EF-G-GTP to EF-G + GDP + Pi -> incoming aminoacyl-tRNA3 (empty tRNA in E site and dipeptide in P site) order of sites in the ribosome during translation - APE elongation factors stay associated with tRNA for msec and allow additional proofreading starting tRNA in P site, empty A site -> releasing energy from bond formation to move into the next position -> uncharged tRNA is released

steps of non-vesicle based protein targeting

step 1 - nuclear protein NLS (nuclear localization signal) binds to importin alpha and beta step 2 - NLS complex travels through the nuclear envelope by a nuclear pore complex step 3 - Ran(Ras-related nuclear protein)-GTP binds to importin beta, importin alpha and NLS gets cleaved step 4 - Ran-GTP binds to alpha and CAS (cellular apoptosis susceptibility protein) -> NLS is released step 5 - complex leaves the nucleoplasm and dissociates into individual components

glycogen

storage homopolysaccharide form of glucose - fuel main storage polysaccharide of animal cells abundant in the liver a polymer of glucose linked by α-1,4 linkage, but has about one α-1,6 linkage per 8-12 residues non-reducing ends and reducing ends when glycogen is used as an energy source, glucose units are removed one at a time from the non reducing ends degradative enzymes can simultaneously work on the non-reducing ends of many branches to speed the conversion of the polymer to the monomer, glucose more branched in comparison to amylopectin - used in glycolysis pathway to liberate energy exists in the alpha configuration

starch

storage homopolysaccharide form of glucose - fuel most important storage polysaccharides in plant cells occurs in two forms - amylose and amylopectin

cellulose

structural component of plants - contains several glucose molecules most abundant organic polymer on earth

cellulose

structural homopolysaccharide form of glucose performs structural roles as opposed to nutritional role and is an important component of the plant cell wall unbranched polymer of glucose linked by β-1,4 linkages exists in the beta conformation - very different function and role! cellulose is formed by lining parallel to each other and connecting through H-bonds - leads to structural roles no role in nutrition because there are no enzymes to cleave the 1,4-glycosidic bonds

catabolism of disaccharides

sucrose and lactose

disaccharides

sucrose, lactose, and maltose carbohydrates formed when two monosaccharides are joined by an O-glycosidic bond - connecting sugar-sugar or sugar-non-sugar moiety (connected by oxygen atom) have polarity defined by the reducing and non reducing ends carbohydrate unit at the reducing end has a free anomeric carbon atom that has reducing activity glycosidic bonds can be cleaved under acidic conditions, but are stable to basic conditions all consumed in the diet - must enter glycolysis to produce energy (metabolism to harvest energy)

RNA polymerase I

synthesizes pre-ribosomal RNA precursor for 28S, 18S, and 5.8S rRNA

first stage of protein synthesis - activation of amino acids

tRNA - carries the specific amino acid single chain with 73-93 ribonucleotides contain 7-15 modified RNA bases, many are methylated or dimethylated, pseudoU 5'-terminus phosphorylated and usually G 3'-terminus (5'-CCA) - activated amino acid is attached to the 3'-OH of A there are 4 stems and 4 loops - anticodon loop consists of seven bases (5' Y-Y--XYZ--R(Y)-N- 3') some residues allow for specific folding of the RNA into the required structure the CCA sequence at the 3' end is the attachment point for the amino acid - A is essential for amino acid linkage in amino acid arm positions two and three on the tRNA form stronger bonds to mRNA for each amino acid there is 1 (or more) tRNA

explanation for codon degeneracy

tRNA(Phe) C-C-A-3' region of tRNA - 3' tRNA link to amino acids 3'-T/C-TT - anticodon region of tRNA that binds to mRNA anticodon region in downwards loop recognizes mRNA codon and brings the correct amino acid to add to the polypeptide

essential components for step 4 of protein synthesis - termination and ribosome recycling

termination codon in mRNA release factors - RF-1, RF-2, RF-3, RRF EF-G IF-3

structure of O, A, and B antigens

the A and B antigens differ only in a sidechain on the terminal sugar the addition of N-acetylgalactosamine or galactose to the O antigen is catalyzed by glycosyltransferases - enzymes that catalyze formation of glycosidic bonds from O -> A or B - attaching a sugar, but a different antigen is added by enzymes genes in a person's DNA code for the specific glycosyltransferases to allow for the addition of antigens A and/or B to the O antigen inside of red blood cell -> lipid bilayer -> carbohydrate chains

N-end rule pathway

the amino-terminal residue has a profound influence on the half lives of many proteins these amino-terminal signals have evidently been conserved during billions of years of evolution the signals are the same in bacterial protein degradation systems and in the human ubiquitination pathway protein stability - conserved during evolution deletion of N-terminal from protein - unable to be expressed

proteoglycans - heparin sulfate and blood coagulation

the blood coagulation pathway is composed of a cascade of proteolytic reactions ultimately generating fibrin thrombin the pro-anticoagulant activity of this cascade is balanced by several natural anticoagulant mechanisms thrombin (essential to coagulation) is inhibited by another blood protein antithrombin that prevents premature blood clotting antithrombin binds to thrombin only in the presence of heparin sulfate - repel each other due to positive charges heparin sulfate increases the binding affinity of thrombin for antithrombin (~2000 fold), thus strongly inhibiting thrombin heparin sulfate bridges the positively charged regions of the two proteins, causing an allosteric change that inhibits thrombin's activity

amylopectin

the branched starch containing glucose residues in α-1,4 linkage, but has about one α-1,6 linkage per 24-30 residues

amino acid code words in mRNAs

the codons for nearly all of the amino acids can be symbolized by XY(AG) or XY(UC) the first two letters of each codon are therefore the primary determinants of specificity start codon - AUG stop codons: 1. UAA - U Are Annoying 2. UGA - U Go Away 3. UAG - U Are Gone

ribosomal RNAs (rRNA)

the constituents and play catalytic roles in ribosome (60% RNA) by reading tRNA and introducing the appropriate amino acids synthesized in the nucleolus and functions as an integral part of the ribosomal machinery used during protein assembly in the cytoplasm many rRNA molecules function as ribozymes - enzymes made of RNA molecules instead of peptides helps catalyze the formation of peptide bonds and is also important in splicing out its own introns within the nucleus

genetic mechanism of SMA

the disorder is caused by a genetic defect in the SMN1 gene, which encodes SMN, a protein widely expressed in all eukaryotic cells (including human cells) and necessary for survival of motor neurons lower levels of the protein results in loss of function of neuronal cells in the anterior horn of the spinal cord and subsequent system-wide atrophy of skeletal muscles mutation: SMN1 pre-mRNA -(splicing)> mRNA -(translation)> normal SMN protein levels SMN2 - polymorphic at exon 7, not usually expressed in normal people protein is very unstable change splicing to increase stable protein product - compensate for loss of SMN1 gene

the wobble hypothesis

the first two bases of a codon in mRNA always form strong Watson-Crick base pairs with the anti-codon, conferring most of the coding specificity the first base of some anticodons (in tRNA, corresponding to 3rd base of codon in mRNA) determines the number of codons read by a given tRNA - the third position in the anticodon can recognize differing numbers of codons on mRNA one codon recognized: 1. anticodon is 3'-X-Y-C-5'; codon is 5'-X'-Y'-G-3' 2. anticodon is 3'-X-Y-A-5'; codon is 5'-X'-Y'-U-3' two codons recognized: 1. anticodon is 3'-X-Y-U-5'; codon is 5'-X'-Y'-A/G-3' 2. anticodon is 3'-X-Y-G-5'; codon is 5'-X'-Y'-C/U-3' three codons recognized - anticodon is 3'-X-Y-I-5'; codon is 5'-X'-Y'-A/U/C-3' X and Y denote bases complementary to and capable of strong Watson-Crick base pairing with X' and Y', respectively wobble bases in the 3' position of codons and 5' position of anticodons 32 tRNAs -> translate all 61 codons -> 21 AAs when an amino acid is specified by several different codons, those codons that differ in either of the first two bases require different tRNAs

functionalities of carbohydrates

the functionalities and capabilities of several proteins and lipids are diversified by attaching sugar (carbohydrate) components - glycoproteins (sugar proteins) and glycolipids (sugar lipids) diverse capabilities upon interacting with other macromolecules coagulation factor III is a cell surface glycoprotein that plays a role in the clotting process by initiating thrombin ABO blood groups are a result of red blood cells coated with specific O/A/B type glycoproteins immune response to pathogenic bacteria is often stimulated by glycoproteins on the surface of bacteria carbohydrates supply details and enhancements to the biochemical architecture of the cell, helping to define beauty, functionality, and uniqueness of the cell

glucose

the most important sugar in the human body found in numerous foods and is the principal sugar that is catabolized (broken down) in a metabolic pathway called glycolysis to release energy glycolysis is the major route of catabolism for carbohydrates the concentration of glucose in the blood is critical to the normal body function and is maintained at a relatively stable concentration (80 to 120 mg/dL) this stable concentration is maintained by two hormones - insulin and glucagon insulin exerts its role in the fed state when concentrations of glucose in the circulating blood is the highest glucagon exerts its role in the fasting state when concentration of glucose in the blood falls down

glucose homeostasis

the process by which insulin and glucagon work to maintain blood glucose levels normal blood glucose level - 80 to 120 mg/dL when BG levels raise - high in fed state, insulin acts to decrease blood glucose through: 1. increasing glycolysis 2. increasing glycogenesis insulin - glucose enters the cell to degrade or forms glycogen to store energy when BG levels fall - low in starving state, glucagon acts to increase blood glucose through: 1. increasing gluconeogenesis - glucose synthesized from non-carbohydrate starting material 2. increasing glycogenolysis - glycogen broken down to glucose

DNA replication

the process of making two daughter strands, where each daughter strand contains half of the original DNA double helix purpose - to conserve the entire genome for the next generation enzymes required - DNA helicase, DNA polymerase raw materials - dATP, dGTP, dTTP and dCTP serve as raw materials occurs in preparation for cell division it requires an RNA primer to start replication products - two daughter strands it involves copying of the entire genome

transcription

the process of synthesis of RNA using DNA as a template purpose - to make RNA copies of individual genes (major difference); RNA makes up 3% of the genome enzymes required - RNA polymerase raw materials - ATP, UTP, GTP and CTP serve as raw materials occurs in preparation for protein translation no primer is required to start!! products - mRNA, tRNA, rRNA and non-coding RNA, like microRNA it involves copying of certain individual genes only

ribosome in protein synthesis

the ribosome is a key player in protein synthesis make up ~25% of dry weight of bacteria ribosome is ~65% rRNA, 35% protein - rRNA forms the core (rRNA is the enzymatic center ) RNA does the catalysis of peptide bond formation - no protein within 18 Å of catalytic site made of two subunits bound together (30S and 50S) in bacteria, with mRNA running through them - 2.7 x 106 kD

glycobiology

the study of the synthesis and structure of carbohydrates and how carbohydrates are attached to and recognized by other molecules such as proteins

amylose

the unbranched starch containing glucose residues in α-1,4 linkage non-reducing end and reducing end

transpeptidase inhibition by beta-latctam antibiotics

the β-lactam region of the drug resembles the D-Ala-D-Ala end of the peptide to which the transpeptidase enzyme binds - competes with the peptide for the active site of the enzyme forms an irreversible bond with the enzyme, blocking cross-linking, so bacteria will not survive the covalent complex irreversibly inactivates the enzyme, blocking synthesis of the bacterial cell wall the absence of protective outer rigid cell wall results in rupture of the inner membrane under osmotic pressure covalent complex - stably derivatized, inactive transpeptidase anti-bacterial activity!

cloning cDNA = mature mRNA

tissue -(isolate RNA)> various RNA-binding proteins -> remove proteins or immunoprecipitate remove proteins -> make cDNA Primer and reverse transcriptase) -> covalently link biotin (B) to cap immunoprecipitate -> remove proteins -> make cDNA Primer and reverse transcriptase) -> covalently link biotin (B) to cap using reverse transcriptase as a tool for biological discovering used to be very hard to find coding sequence of genes (exons) - targeting mature RNA and finding 5' and 3' ends (full coding sequence) clone cDNA out protein specific binding - immunoprecipitate poly T recognizes poly A tail and uses reverse transcriptase and primer to form hybrid need to know full length of cDNA to be useful - use 5' cap of high energy and link biotin to cap fully finished - no gap (ssRNA - PCR not finished) degrade rna that aren't paired to cDNA - isolate biotin linked molecules and wash away no biotin on molecules - only fully transcribed pieces are retained digesting RNA and sequencing dsDNA - full sequence of gene

transcription factors

transcription factors are transcription-activating proteins that search the DNA looking for specific DNA-binding motifs transcription factors tend to have two recognizable domains: 1. DNA-binding domain 2. transcriptional activation domain

summary of DNA-dependnent synthesis of RNA

transcription is catalyzed by DNA-dependent RNA polymerases, which use ribonucleotide 5'-triphosphates to synthesize RNA complementary to the template strand of duplex DNA - transcription occurs in several phases: binding of RNA polymerase to a DNA site called a promoter, initiation of transcript synthesis, elongation, and termination bacterial RNA polymerase requires a special subunit to recognize the promoter - as the first committed step in transcription, binding of RNA polymerase to the promoter and initiation of transcription are closely regulated transcription stops at sequences called terminators eukaryotic cells have three types of RNA polymerases - binding of RNA polymerase II to its promoters requires an array of proteins called transcription factors elongation factors participate in the elongation phase of transcription - the largest subunit of pol II has a long CTD, which is phosphorylated during the initiation and elongation phases

key concepts of transcription - promoter

two consensus sequences at −10 (TATAAT) upstream from beginning of RNA replication and −35 (TTGACA) recognized by RNA polymerase for σ^70 subunit binding - called TATA sequences ("TATAbox") A-T rich upstream promoter (UP) element between −40 and −60 binds the α subunit A-T rich sequences promote strand separation these sequences and their spacing govern efficacy of RNA polymerase binding and therefore affect gene expression level E. coli - transcription initiation signal promoters typical E. coli promoters recognized by an RNA polymerase holoenzyme - different σ subunits allow for transcription of various sequences UP - upstream promoter that strongly stimulates transcription; not in all genes, increases expression levels of genes RNA polymerase binds to a sequence called the promoter to begin transcription - generates positive supercoils ahead, later relieved by topoisomerases

E3

ubiquitin ligase enzyme most important - high affinity and specificity to the substrate transfers ubiquitin to substrate as a label hundreds of these enzymes responsible for down expression of a protein in the cell

E1

ubiquitin-activating enzyme

E2

ubiquitin-conjugating enzyme becomes donor of ubiquitin

tRNA(fMet)

uncharged tRNA specific to fMet without fMet linked

housekeeping gene

under constitutive expression - constitutive promoter constantly expressed in ~all cells - structural proteins of chromosomes, RNA polymerases, DNA repair enzymes, ribosomal proteins, enzymes involved in glycolysis and other basic metabolic processes, and many of the proteins that form the cytoskeleton must be expressed - structure to support function, etc.

reducing property of glucose

used as one of the earlier tests in the clinical estimation of circulating glucose in the blood Fehling's solution - control is blue, positive test is red glucose (an aldehyde) + 2Cu2+ (blue, oxidized form) -(OH-)> gluconic acid + Cu2O (cuprous oxide, reduced form, red) the amount of Cu2O formed is proportional to the amount of glucose present in the urine from left to right the test tubes contain no sugar (control), 0.1% glucose solution (cloudy blue), 1% glucose solution (orange), 10% glucose solution (brown) diagnostic test for diabetes - high blood glucose -> excreted in urine glucose (reducing agent) reacts with copper, forming cuprous oxide - extrapolate the amount of glucose in blood circulation

the genetic code is universal, with a few exceptions

used by prokaryotes and eukaryotes, across species there is little room for variation in the genetic code mitochondria have a small genome (13 proteins, 2 rRNAs, and 22 tRNAs) and use a slightly different code: 1. UGA encodes Trp in vertebrate mtDNA, instead of STOP 2. AGA/AGG encodes STOP in vertebrate mtDNA, instead of Arg the genetic code is degenerative and universal in all species with minor deviations in mitochondria

common sequences in promoters recognized by eukaryotic RNA polymerase II

various regulatory sequences - TATA box and Inr initiator sequence many of the regulatory sequences are within a few 100's bp of the TATA box on the 5′ side, others may be 1000's away RNA polymerase I (5.8S, 18S, 28S rRNA) - upstream polymerase element and core promoter element RNA polymerase II (mRNA) - various regulatory sequence, TATA box, and initiator (~8 bp of DNA is unwound) - chicken ovalbumin, adenovirus late, rabbit beta globin, mouse beta globin major RNA polymerase III (tRNAs, 5S rRNA)

example of aggrecan

when your foot hits the ground while walking, pressure is exerted, squeezing water from the glycosaminoglycans and cushioning the impact when pressure is released, water rebinds - water binding and releasing mechanism for compression osteoarthritis can result from proteolytic degradation of aggrecan and collagen in the cartilage!!