bio chem quiz 7

what is heparin and heparan sulfate?

• heparin is a linear polymer

• heparan sulfate is heparin-like polysaccharide but attached to proteins

• highest negative charge density biomolecules

• prevent blood clotting by activating protease inhibitor antithrombin

• binding to various cells regulates development and formation of blood vessels

• can also bind to viruses and bacteria and decrease their virulence

what is the molecular basis of heparin enhancement of the binding of thrombin to antithrombin?

• heparin is negatively charged and acts as a tether to bring proteins together and inhibit thrombin

• heparin connects thrombin to its inhibitor, antithrombin, then prevents blood clots from forming

• heparin is an example of a GAGs: a polysaccharide that is negatively charged and has 2 repeating disaccharides (2 repeating sugars)

what is a glycoprotein as a glycoconjugate?

• a glycoprotein is a carb attached to a protein

• a protein with small oligosaccharides (3-10 carbs long) attached

  • carbohydrates attached via its anomeric carbon to amino acids on the protein

    • common connections occur at Ser, Thr, and Asn

  • about half of mammalian proteins are glycoproteins

  • only some bacteria glycosylate a few of their proteins

  • carbohydrates play a role in protein-protein recognition

  • viral proteins are heavily glycosylated; this helps to evade the immune system (tricks immune system into thinking its our protein)

what are the two types of glycosidic bonds?

1) O-linked : attaching a carb to oxygen; glycosidic bond to the hydroxyl group

2) N-linked : attaching a carb to nitrogen; N-glycosyl bond

what do glucotransferases do?

glucotransferase are enzymes that recognize amino acids and attach carbohydrates

what are glycolipids as a glycoconjugate?

• glycolipids are lipids with covalently bound oligosaccharide

  • they are parts of plant and animal cell membranes

  • in vertebrates, ganglioside carbohydrate composition determines blood group

  • in gram negative bacteria, lipopolysaccharides cover the peptidoglycan layer

  • often used for cell-cell recognition by changing oligosaccharides attached to the membrane

what are proteoglycans as glyconjugates? and what are the 2 main types?

• proteoglycans are GAGs attached to proteins (that are usually attached to cells or extraceullar matrix)

• sulfated glucoseaminoglycans attached to a large rod-shaped protein in cell membrane

  • syndecans: protein has a single transmembrane protein

  • glypicans: protein is anchored to a lipid membrane

  • interact with a variety of receptors from neighboring cells and regulate cell growth

what is the interaction of cells with the ECM?

interaction of the cells with ECM:

• some internal membrane proteins are proteoglycans

  • syndecans

• other integral membrane proteins are receptors for extracellular proteoglycans

  • integrins

  • integrins are a class of receptors that feel the matrix and cells around them (feeling micro-enviornment)

  • integrins interact with syndecans

• these proteins link cellular cytoskeleton to the ECM and transmit signals into the cell to regulate: cell growth, cell mobility, apoptosis, and wound healing

what is the cell signaling mechanism of integrans?

→ integrins interact with the extracellular matrix primarly through the syndecans/proteoglycans, which information can get relayed internally to rearrange intracellular cytoskeleton (mechanism of how cells feel their microenvironment through integrins)

→ integrins have a specific binding site to allow the proteoglycans to attach to them. the integrin then changes its conformation and becomes more linear. structural change gets induced into the intracellular cytoskeleton, which will rearrange or induce a signal

what is lectin?

lectin are proteins that specifically bind carbohydrates to help with cell recognition

what are the two methods of analysis for glycoconjugates?

1) global profiling: all types of oligosaccharides on a kidney cell

2) targeted structure analysis: oligosaccharides in a specific protein/lipid

how to use MS for analysis of glycoconjugates / how to interpret it?

positioning of the m/z peaks = the type of oligosaccharide

height of the peak = abundance / how much

what are the basics of nucleotides and nucleosides?

• nucleotide =

  • nitrogeneous base (pyrimidine: smaller ring or purine: larger ring)

  • pentose sugar

  • phosphate

• nucleoside =

  • removal of phosphate group

  • nitrogeneous base

  • pentose sugar

• carbons AND nitrogen atoms on the nitrogeneous base are numbered in cyclic format

• carbons of the pentose are designated N’ to alleviate confusion

• nitrogen 1 on pyrimidine and nitrogen 9 on purine are used to connect to a sugar

what are the functions of nucleotides and nucleic acids?

• nucleic acids are polymers of nucleotides used for (polymeric form):

  • storage of genetic info (DNA)

  • transmission of genetic info (mRNA)

  • processing of genetic info (ribozymes)

  • protein synthesis (tRNA and rRNA)

• nucleotides are also used in the monomer form for cellular functions (monomeric form):

  • energy for metabolism (ATP)

  • enzyme cofactors (NAD+) (Nicotinamide adenine dinucleotides: used to store electrons)

  • signal transduction (cAMP)

how do pentose forms differ in some nucleic acids and nucleotides?

how pentose forms differ:

• beta-d-riboduranose in RNA (has a 2’ OH group)

• beta-2’-deoxy-d-ribofuranose in DNA (does not have a 2’OH group, has a hydrogen on 2’ position)

• different puckered coformations of the sugar ring are possible

how to tell endo and exo pucker conformations? which do RNA and DNA typically have?

endo conformation = if carbon 2 or 3 is on the same side as the 5’ carbon

exo conformation = if carbon 2 or 3 is on the opposite side (pointing away) as the 5’ carbon

DNA typically takes on a 2’ endo conformation and RNA typically takes on a 3’ endo conformation

what are nitrogenous bases?

• nitrogenous bases are derivatives of pyrimidine (smaller ring) or purine (larger ring)

• nitrogen containing heteroaromatic molecules

• planar or almost planar structures (sp2 hybridization makes the ring flat)

• absorb UV light around 250-270 nm (because of conjugated ring system; if you hit with light ~260 nm the amount of light absorbed can be used to determine the concentration)

• cytosine, adenine, and guanine are found both in DNA and RNA

• thymine is only found in DNA

• uracil is only found in RNA

• all are good H-bond donors and acceptors (key structural feature to form different H-bonds)

• neutral molecules at pH 7

how many hydrogen bonds can the following form based on structure: cytosine, thymine, and uracil?

cytosine: 3 H bonds

thymine: 2 H bonds

uracil: 2 H bonds

what is the nucleoside naming for the following deoxyribonucleotide structures

1. deoxyadenosine

2. deoxyguanosine

3. deoxythymidine

4. deoxycytidine

what is the nucleoside naming for the following ribonucleotide structures

1. adenosine

2. guanosine

3. uridine

4. cytidine

what is the beta-N-glycosidic bond?

• in nucleotides, the pentose ring is attached to the nitrogenous base via a N-glycosidic bond (connects a ribose sugar to nitrogenous base)

• the bond is formed to the anomeric carbon of the sugar in beta configuration

• the bond is very stable and helps to give DNA its high stability

• the bond is formed:

  • to position N1 in pyrimidines

  • to position N9 in purines

• this bond is quite stable toward hydrolysis, especially in pyrimidines

• bond cleavage is catalyzed by acid

• in monomeric form, the bond can freely rotate. when connected in strands, it can take on particular angles

  • syn = on top of each other

  • anti = opposite

what is the tautomerism of nitrogenous bases?

• prototropic tautomers are structural isomers that differ in the locations of protons

• keto-enol tautomerism is common in ketones

• ketone form is more stable at pH 7 (favors ketone form), by changing the pH you can shift to favor the enol form

• Lactam-lactim tautomerism occurs in some heterocycles

• both tautomers exist in solution, but the lactam (ketone) forms are predominant at neutral pH

what happens as you change the pH from lactam to lactim?

at lactam, the pH is neutral. as the pH decreases: lactam → lactim → double lactim

changing lactam-lactim form disrupts the hydrogen bonding when holding the nucleotide strands together → this causes weakening of the structure for RNA and DNA

what are minor nucleosides in DNA?

minor nucleosides:

• modification is done after DNA synthesis

• 5-methylcytosine is common in eukaryotes and is also found in bacteria (methylation of DNA creates gene suppression)

• N6-methyladenosine is common in bacteria but not found in eukaryotes

• epigenetic marker:

• way to mark own DNA so that cells can degrade foreign DNA (prokaryotes)

• way to mark which genes should be active (eukaryotes)

• could the environment turn genes on and off in an inheritable manner? (epigenetic inheritance: modify DNA to determine the degree of expression of genes = epigenetic markers determine by the environment; epigenetic markers = modified nucleosides)

what are polynucleotides?

polynucleotides:

• covalent bonds are formed via phosphodiester linkages (to connect nucleotides together)

  • negatively charged backbone

• DNA backbone is fairly stable (because the linkage is very stable)

  • hydrolysis accelerated by enzymes (DNases: recognize sequence to cut phosphodiester linkage)

• RNA backbone is unstable

  • in water, RNA lasts for a few years

  • in cells, mRNA is degraded in a few hours (vaccine); has a high turnover in the cell

• linear polymers

  • no branching or cross links

• directionality

  • the 5’ end is different from the 3’ end

  • we read the sequence from 5’ → 3’

  • enzymes read / sequence based on this directionality

what is the hydrolysis of RNA?

hydrolysis of RNA:

• RNA is unstable under alkaline (high pH) conditions

• hydrolysis is also catalyzed by enzymes (RNases)

• RNase enzymes are abundant around us

  • S-RNase in plants prevent inbreeding by preventing pollen tube formation

  • RNase P is a ribozyme (enzyme made of RNA) that process tRNA precursors

  • Dicer is an enzyme that cleaves double stranded RNA into oligonucleotides

    • can be used to target specific sequences (RNA interference tech)

    • protection from viral genomes

in terms of the mechanism of base catalyzed RNA hydrolysis, what is the role of OH-

the 2’ OH- allows for cleavage of the backbone to create 2 separate nucleotide groups

what are the hydrogen bonding interactions observed?

• two bases can hydrogen bond to form a base pair

• for monomers, a large number of base pairs is possible

• in polynucleotide, only a few possibilites exist (only specific base pairing to give complementary hydrogen bonding)

• watson-crick base pairs predominate in double stranded DNA

  • A pairs with T : 2 H bonds

  • C pairs with G : 3 H bonds

• purine pairs with pyrimidine (Chargaff’s rule: 1:1 ratio of purine to pyrimidine)

basics of AT and GC base pairing?

→ the base pairing of A and T occurs by 2 hydrogen bonds

→ the base pairing of G and C occurs by 3 hydrogen bonds

→ if you increase the number of hydrogen bonds = base pairs get closer together

• bacteria growing under hot conditions = more GC base pairing to hold the strands together. colder = more AT base pairing to allow DNA to open up for replication

what is the watson-crick model of B-DNA

B DNA is the most common form

uneven in grooves = major and minor grooves

it is 3.4A as the distance from one base to another on the same strand

base stacking increases the van der waals forces, increasing the stability of the DNA strand

more base stacking = less light absorbed

what are the 2 other forms of DNA besides B DNA?

A form: forms by dehydration of B form, wider diameter and shorter length; shorter, wider, found in dehydrated conditions

Z form: stretching and now a left handed helix; long, skinny, left handed, bases are exposed, found in sites where DNA is being copied

what are some ways to see structure differences in the B, A, and Z form of DNA?

helical sense diameter, base pairs per helical turn, helix rise per base pair, base tilt normal to the helix axis, sugar pucker conformation, and glycosyl bond conformation

what is the replication of genetic code?

for the replication of genetic code:

• strand separation occurs first

• each strand serves as a template for the synthesis of a new strand

• synthesis is catalyzed by enzymes known as DNA polymerases

• a newly made DNA molecule has one daughter strand and one parent strand

what is messenger RNA?

messenger RNA is the code carrier for the sequence of proteins

• is synthesized using DNA template and generally occurs as a single strand

• contains ribose instead of deoxyribose

• contains uracil instead of thymine

• one mRNA may code for more than one protein

  • two assumptions: (1) a single RNA strand will make multiple proteins and (2) selection of different exons to create different versions of the protein (alternative splicing)

• together with transfer RNA (tRNA), transfers genetic information from DNA → proteins

what is the difference between monocistronic and polycistronic gene strands?

monocistronic: RNA codes for a protein for an individual gene; eukaryotic mRNAs are monocistronic; allows for fine tune regulation of proteins (higher regulation) and tissue differentiation to give them their own phenotypes

polycistronic: single mRNA has multiple genes that can be translated; prokaryotic individuals like bacteria and archaea have this type

what are palindromes?

palindromic sequences can form hairpins and cruciforms (secondary structures for DNA and RNA)

palindromes: words or phrases that are the same when read backward or forward

ex: civic, racecar

what is the difference between a palindrome and a mirror repeat? how does a hairpin form?

palindrome: traditional; think what the complementary strand looks like, complementary strand should read the same

mirror repeat: rotate 180° on y axis, if it reads the same sequence = mirror repeat

hairpin: palindrome sequence that folds in on itself; hairpins come together to form a cruciform

how do RNA molecules have quite complex structures?

RNA molecules can have quite complex structures, such as RNase P. multiple hairpins build on one another, which results in a lot of non-Watson and Crick base pairs in more complex structures

what are complex structures stabilized by?

complex structures are stabilized by non-watson and crick base pair interactions

• the rule breaking of Watson-Crick gives ribozymes unique structures and function

  • ex: tRNA, hammer head ribozyme used to degrade mRNA, and intron (ribozyme) catalyzes its own excision from between exons in an mRNA strand

what is deamination?

deamination is a molecular mechanism of spontaneous mutagenesis

• spontaneous: happens due to equilibrium shift of structures

• deamination: hydrolysis rxn, amine group is replaced by a carbonyl group

  • very slow reactions and unfavorable

  • large number of residues

  • the net effect is significant: 100 C→U events/day in mammalian cells

• repaired through base excision repair pathways (BER): recognizes non-Watson-Crick base pairs and replaces them with the correct one

what is depurination?

depurination is a molecular mechanism of spontaneous mutagenesis

• depurination: breakage of N-glycosidic bond

  • N-glycosidic bond is hydrolyzed

  • significant for purines: 10,000 purines lost/day in a mammalian cell

what are the two mechanisms for which external factors induce mutations?

molecular mechanisms of oxidative and chemical mutagenesis

1. oxidative damage

   1. creates ROS (superoxide, hydrogen peroxide, and hydroxyl radical): ROS is way more reactive and may react with biological material

   2. hydroxylation of guanine

   3. mitochondrial DNA is most susceptible

2. chemical alkylation

   1. add carbon chains/groups to bases

   2. methylation of guanine

   3. cells have mechanisms to correct for most of these modifications but must do so with the correct timing

what are alkylating agents?

alkylating agents donate carbon groups to DNA and RNA

what is radiation-induced mutagenesis?

molecular mechanism of radiation-induced mutagenesis:

• UV light induces dimerization of pyrimidines (causes covalent linkage and a change in the phosphodiester backbone); this may be the main mechanism for skin cancers

• ionizing radiation (x rays and gamma rays) causes ring opening and strand breaking → these are difficult to fix

• cells can repair some of these modifications but others cause mutations. accumulation of mutations is linked to aging and carcinogens

how can nucleotides function as an energy source?

energy source of nucleotides:

→ changing the base will give different types of energy carriers (swap out bases to give different metabolic pathways)

• protein synthesis (ribosome function) and signal transduction (G-proteins)

• phospholipid and membrane synthesis

• carbohydrate/glycogen synthesis

how can nucleotides function as coeznymes?

coenzymes:

• carries acyl groups (to start citric acid cycle)

• electron carriers (are modified nucleotides and have a conjugated double bond)

how can nucleotides function as regulatory molecules?

regulatory molecules:

• cell signaling and signal transduction pathways

what is recombinant DNA?

• recombinant DNA is artificially created DNA that combines sequences that do not occur together in nature '

• basis of much of modern molecular biology

  • molecular cloning of genes: gene of one organism is put into another organism

  • overexpression of proteins

  • transgenic food, animals

what is organism cloning? DNA cloning and its steps?

organism cloning

• creation of genetically identical copies of an organism

• organisms may not appear identical due to regulation of gene expression during development

DNA cloning

• creation of identical copies of a piece of DNA (gene)

• isolate a specific gene from the source organism and amplify it in the target organism

basic steps

• cut the source DNA at the boundaries of the gene

• select a suitable carrier DNA (vector)

• insert gene into the vector

• insert the recombinant vector into host cell

• let the host cell produce multiple copies of recombinant DNA

what is step 1 to DNA cloning?

step 1: generate recombinant vector

• goal: gene from one organism into a plasmid DNA

• cloning vector is cleaved with restriction endonuclease

• DNA fragment of interest is obtained by cleaving chromosome with a restriction endonuclease

• fragments are ligated to the prepared cloning vector

• recombinant vector (plasmid) made

what is step 2 to DNA cloning?

step 2: introduce DNA into organism (transformation)

• DNA introduced into host cell

• propagation (cloning) of transformed cell produces many copies of recombinant DNA

what are restriction endonucleases?

restriction endonucleases are enzymes that target palindromic sequences of DNA

• palindromic sequences in DNA are recognized by restriction enzymes. sequences

• common in bacteria

  • eliminates infectious viral DNA

• some make staggered cuts

  • sticky ends

• some make straight cuts

  • blunt ends

• large number are known

  • commerically available

  • well documented: REBASE

what are DNA ligases?

DNA ligases are enzymes that covalently joins two DNA fragments

• it recognizes the phosphodiester backbone and ligases it together, not sequence specific

• normally function in DNA repair

• human DNA ligase uses ATP

• bacterial DNA ligase uses NAD

• energy dependent