Nucleotides and nucleic acids

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Biochem 1 Chapter 10 Test 3

Biological molecules that possess heterocyclic nitrogenous basas as principal componetns of its structure?

nucleotides and nucleic acids

Biochemical roles of nucleotides

-intermediate cell metabolism
-nucleic acid is the element of heredity
-agent of genetic information transfer
-its nucleotides residue sequence encode protein

Two basic kinds of nucleic acid

DNA, RNA

complete hydrolysis of nucleic acid gives what as products?

liberates nitrogenous base
five sugar carbon
phosphoric acid... all in equal amount

DNA sugar

2-deoxy-ribose

RNA sugar

ribose

DNA stores genetic info while RNA transcript and translates it, except?

when regarding virus. some virus caries their genetic code in their RNA

pyrimidine

-single ring, 2 N atoms
-aromatic, planar
-number clockwise
-cytosine, thymine, uracil
-found in DNA, RNA
-bound to sugar and phosphate

purine

-double ring nitro base, 9 atoms
-combination of pyrimidine+imidazole
-not planar, pucked btw rings
-found on DNA, RNA
-adenine, guanine

pyrimidine+imidazole

purine

thymine

-2-oxy-4-oxy-5 methyl pyrimidine
-only found on DNA
-The complementary base to adenine

uracil

-2-oxy-4-oxy-pyrimidine
-nitrgenous base found only in RNA; base pairs with adenine (it replaces the Thymine in DNA during transfer)

cytosine

-2-oxy-4-amino pyrimidine
The nitrogenous base that makes three hydrogen bonds with guanine.

pyrimidine derivative

dihydrouracyl

purine derivative

hypoxanthine, xanthine(rarely found)
-uric acid (never found in nucleic acid)

uric acid

most oxidized state of purine, never found in nucleic acid

Who had the pairing date of DNA but didnt understand its implications?

Erwin Chargaff

Where base pairs in DNA double helix arises from?

hydrogen bonds

Responsible for X-ray of DNA fiber diffraction data

Rosalind Franklin

Who discover double helix?

Francis Crick

who figured out H-bonds on DNA?

James Watson

Tautomerism

-refers to equilibrium of two different structures of the same compound.
-usually differs at the point of attachment of a hydrogen atom
-most common form is keto-enol form

why keto-enol tautomerism shift favors keto (lactam)?

-Solvent polarity is also very important in determining the magnitude of Ke. The enol form is less polar than the keto tautomer because of the intramolecular hydrogen bonding. Thus, an increase in solvent polarity favors the more polar keto form.

Why are they called bases?

Because N1 and N3 of pyrimidine, and N1, N3, and N7 of purine can accept protons.

What's the sugar/base link?

an N-glycosidic bond btw C1' of sugar and N1 or pyrimidine, or N9 of purine

What gives bases the capacity to undergo tautomerism?

-aromaticity
-electro-rich nature of -OH and -NH2
-pyrimidines and purines exist as tautomeric (one is leto the other enol)

Lactam

-keto form of purine and pyrimidine
-predominates at neutral pH

Lactim

eno form of purine and pyrimidine

pKa value relevance

These pKa values specify whether hydrogen atoms are associated with the various ring nitrogens at neutral pH. As such, they are important in determining whether these nitrogens serve as H-bond donors or acceptors. One base would have a N-1 with pKa of 9.5, N-3 of 5, meaning the base will be forming the bond.

Keto-enol Tautomerism rxn

What participates in H-bond formation on DNA?

the amino groups of cytosine, adenine, and guanine; the ring nitrogens at position 3 of pyrimidines and position 1 of purines; and the strongly electronegative oxygen atoms attached at position 4 of uracil and thymine, position 2 of cytosine, and position 6 of guanine

cytosine-guanine bond

adenine-thymine

Numbering purine and pyrimidine

Which base does not undergo tautomerism?

adenine

What 2 forces hold DNA structure?

hydrogen bonds between complementary base pairs inside the helix and the Van der Waals base-stacking interaction.

why is it more difficult to separate DNA strands that contain more G-C pairs than A-T pairs?

because C-G contain 3 H-bonds while A-T only 2

Numbering ribofuranoses

-When these ribofuranoses are found in nucleotides, their atoms are numbered as 1', 2', 3', and so on to distinguish them from the ring atoms of the nitrogenous bases.
-the seemingly minor difference of -OH group at the 2'-position has far-reaching effects on the 2nd structures available to RNA and DNA, as well as their relative susceptibilities to chemical and enzymatic hydrolysis.

Nucleosides

-base+sugar by N-glycosidic bond
-anomeric carbon of sugar

N-glycosidic bond

anomeric C-1' to N-1 or pyrimidine or N-9 or purine
-always beta conformation in nucleoside

Namining nucleoside

Nucleosides are named by adding the ending -idine to the root name of a pyrimidine or -osine to the root name of a purine.
-The common nucleosides are thus cytidine, uridine, thymidine, adenosine, and guanosine

inosine

-nucleoside formed by hypoxanthine+ribose
Inosine is commonly found in tRNAs and is essential for proper translation of the genetic code in wobble base pairs. it most closely resembles guanine and it can base pair with either A, U, or C which gives the tRNA's more flexibility.

Nucleoside and nucleotides conformation

--exist in syn or anti
-syn pyrimidine, the -O at C-2 lies right about furanose ring
-anti pyrimidine, steric interference is avoided (favored)
-purine can be either syn or anti; either way base and ring are not coplanar, lie approximately 90° to one another

Nucleoside solubility

-more soluble than free base due to hydrophilicity of sugar
-stable alkali
-pyrimidine nucleoside resistant to acid hydrolysis
-purine nucleoside easly hydrolyzed in acid to yield free base and pentose

Nucleotides

-when phosphoric acid is esterified to a sugar -OH group of a nucleoside.
-OH group avail. in ribose ring C-2',C-3', C-5'
-OH group avail in d-ribose ring C-3', C-5'
-majority of monomeric nucleotides in cell are ribonucleotides

Ribonucleotides

-nucleotides having 5'-phosphate groups
-majority of monomeric nucleotides in cell
-four common ones: AMP, GMP, CMP, UMP

AMP

-adenosine 5'-monophosphate
-5'-AMP
-adenylic acid

GMP

-guanosine 5'-monophosphate
-5'-GMP
-guanylic acid

CMP

-5'-CMP
-cytidylic acid
-cytidine 5'-monophosphate

UMP

-5'-UMP
-uridylic acid
-uridine 5'-monophosphate

3'-AMP

there can be 3'-NMP as well as 2'-NMP, where N is a generic designation for nucleoside.
-do not occur naturally
-biochemically important as products of polynucleotide or nucleic acid hydrolysis

Nucleic acid acidity is due to...

-pKa for 1st dissociation of a proton from phosphoric acid moiety is 1.0 or less
-pK2 value for second dissociation is 6.0. So at neutral pH and above, the net charge on a nucleoside monophosphate s -2
-nucleic acid are polymers of nucleoside monophosphate, derive their name from the acidity of their phosphate group.

Cyclic Nucleotides

-nucleoside monophosphates in which the phosphoric acid is esterified to 2 of availible ribose -OH group
-found in all cells
-cAMP, cGMP important regulators of cellular metabolism

NDP and NTP

-addition of phosphate groups to phosphoryl group of nucleotide through formation of phosphoric anhydride linkage
-AMP>>ADP>>ATP
-groups are α,ß,ϒ
-linkage readily hydrolyzed by acid, liberating inorganic PO3- (P_i))and NMP
-α the group bound to pentose
-occur in the free state in cell
-for deoxy: dAMP>>dADP>>dATP
-there is uracil derivatives for DNA but not Thymine derivative for RNA

Polyprotic acids

-ionize in steps, have more than 1 hydrogen, donate 1 proton at a time
-a subtance capable of ionizing more than one proton in water
-NDP's and NTP's are relatively strong, dissociating 3-4 protons
-form stable complex with cations Mg2+ and Ca2+
-since Mg2+ is

NDPs and NTPs Mg2+ complex

-after dissociating protons, phosphate anion on nucleotide form stable complexes with Mg2+ and Ca2+.
-because there is hight intracellular Mg2+ concentration, those complexes occurs primarily as Mg2+

Testing for NDP and NTP

-quantitative liberation of Pi upon treatment with HCL at 100°C for 7 min.

Nucleoside 5'-Triphosphates Functions

-indispensable agent in metabolims due to phosphoric anhydride bonds they possess, they are prime source of chemical energy to do biological work
-ATP, GTP, UTP,CTP
-all four NTPs and their dNTPs are substrate for synthesis of nucleic acid

ATP

(adenosine triphosphate) main energy source that cells use for most of their work

GTP

A nucleotide composed of guanine, ribose, and three linked phosphate groups. It is incorporated into the growing RNA chain during synthesis of RNA and used as a source of energy during synthesis of proteins

UTP

Activation of sugars in biosynthesis of CHO and glycoproteins.

CTP

Activation of reactants in phospholipid biosynthesis.

Nucleotide's base function

-division of labor
-information symbol, telling what metabolic fxn each nucleotide should do
-biochem reaction uses phosphate or pyrophosphate group transfer, but sugar and base are left out of it
-info symbol extends to nucleic acids, where it serve as info symbol for genetic info.

Nucleic Acid

-linear polymer of nucleotides linked 3'-5' by phosphodiester bridges
-originally formed as 5'-nucleoside monophosphate and successively added to the 3'-OH of preceding nucleotide (giving polymer direction sense).
-two major classes: DNA, RNA

Shorthand notation

-a way of drawing bonds by substituting a single line for each single covalent bond
-backbone of nucleic acid ,vertical line is the furose, diagonal the PO3- bridge
-5'-3' direction
-the diagonal slash rum from middle to bottom, the middle 3' and bottom 5'-C

Shorthand diagram

Base Sequence

-the only variation in nucleic acid structure
-give polymer unique identity
-lowercase"p" used to indicate PO3- bridges btw bases, but rarely used, except on the ends
-"p" on beginning of sequence means PO4 on 5' end
-"p" on the end of sequence means PO4 on 3'-OH end
-to distinguish RNA from DNA just place "d" ex: d-AAGT

How many "chromosomes" does E.coli has?

-one
-circular
-2.9x10^9 D

Where is DNA found in eukaryotic cells?

-principally in 2 copies in the diploid chromosomes of the nucleus
-also occurs in mitochondria, chloroplast (where it encodes proteins and RNAs unique to those organelles.

RNA types

-mRNA, tRNA, siRNA, rRNA
cells contain up to 8x more RNA than DNA
-siRNA only found in eukaryotic cells

DNA double helix

-"base pairs" arises from -H bonds
-Edwin Chargaff rule: [A]=[T] and [C]=[G]
-Rosalind Franklin: x-ray fiber diffraction
-Watson and Creek: complementary double strand helix; genetic replication

DNA structure

-anti parallel double helix
-diameter of 2 nm
-DNA size represented by # of nucleotides base pair
-length of 1.6 million nm in E. coli
-compact and folded, very long and easily shredded into shorter fragments during isolation
-eukaryotic DNA wrapped around histone to form nucleosomes
-base pairs A:T, C:G

canonicals

A:T, C:G base pairs are referred as canonical
-canonical comes from kanon = rule

DNA in form of Chromosome

-the bigger the x-some, the more protein associated to it
-phosphate group on backbone interact ionically to histone to form nucleosomes
-contain other proteins so called non-histone x-somal protein

histone

-arginine and lysine rich basic protein
-interact ionically with anionic phosphate group in DNA
-forms nucleosomes (compact DNA)
-found in eukaryotes only as protein core
-role in gene regulation
-pairs of 4 different histone polypeptide
.

nucleosome

The basic, beadlike unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound around a protein core composed of two copies of each of four types of histone.

DNA picture perfect

non-histone chromosomal protein

involved in regulating which genes in DNA are transcribed at any given moment

mRNA in Eukaryotes

-DNA- like RNA
-A type of RNA, synthesized from DNA, that attaches to ribosomes in the cytoplasm and specifies the primary structure of a protein.
-first synthesized into large precursor in the nucleus called heterogeneous nuclear RNA, hnRNA.
-introns, exons and contain poly(A) tails
-alternative slicing is 50% or more

(T/F)Eukaryotes mRNA encodes only one polypeptide at a time, while prokaryotes mRNA may contain info for synthesis of many proteins

True

When is mRNA synthesized?

transcription

Transcription

-process whereby the DNA sequence in a gene is copied into mRNA
-only genetic unit in DNA is transcribed to mRNA
-synthesis of mRNA goes from 5'-3' direction, meaning it uses single strand of DNA going from 3'-5.
-RNA polymerase II process it, methylating 5' end

poly(a) tail

-After an mRNA is transcribed from a gene, the cell adds a stretch of A residues (typically 100-200) to its 3' end.,
-(3' end) protects against exonucleases
-also found in hnRNA
-adenylic acid residues
-stability

introns

A non-coding, intervening sequence within a eukaryotic gene that gets cut out of the RNA molecule before the RNA becomes functional.
-cut by siRNA
-a.k.a intervening sequence

splicing

The process of removing introns and rejoining cut ends of exons, forming mature mRNA

ribosome

-2/3 RNA, 1/3 protein
-each subunit has one or more piece of RNA and a number of proteins
-most abundant organelle in the cell
-big+small unit, dissociate from each other if Mg 2+ [ ] is below
10^-3M
-small bind to mRNA
-big unit binds to tRNA during translation, providing peptidyl transferase activity
-size given by Svedbeg (S) unit (sediment centrifugation)

23S rRNA in E.coli

peptidyl transferase, not a protein

rRNA

-scarfold for ribosomal protein
-its process takes place in the nucleus
-65% of RNA in ribosome is rRNA

Prokaryote subunit

e.coli ribosomal subunit has a sedimentation coefficient of 30S and 50S
-30S of ecoli contain a single RNA chain and the large subunit has two rRNA

Eukaryote subunit

larger than prokaryotic ribosomes, 40S and 60S

rRNA modified nucleotides

pseudouridine, ribothimydylic acid, methylated bases

rRNA modified base structure

tRNA

-small polynucleotide chains
-several bases usually methylated
-each a.a. has at least one unique tRNA
-3'-terminal sequence is always CCA-a.a.
-aminoacyl tRNA are substrate of protein synthesis
-5 tRNA can carry Leu
-contain anticodon

siRNA

-small inhibitory RNA, fragments of RNA that inhibit transcription, but degradation of mRNA
-post transcriptional gene silencing
-dsRNA highly homologous to RNA strand
-duration of knockdown last 7-10 days
-can transfer to daughter cell

Where does transcription takes place?

nucleus

Where does translation takes place?

cytoplasm

(T/F) Bacteria transcription and translation happens at the same time

True

RNA editing

-change mRNA sequence encoded by gene
-more than 50% of sequence can be altered
-insertion, deletion, and conversion of nucleotides
-guide RNA are bound with "modules" of preedited mRNA

tRNA structure

-74 to 95 nucleotides
-unique due to modified bases
-change made by tRNA-modifying enzyme
-intracellular H bond
-cloverleaf structure
-cleavage modified anticodon
-3' end -CCA

Charged tRNA

-need aminoacyl-tRNA as a key to specificity
-carboxy group of amino acid attach to adenine nucleotide on the tRNA end -CCA
-need ATP

tRNA structure (image)

aminoacyl-tRNA

-key to specificity of amino acid and its tRNA
-its an enzyme
-there are 20 of them, one for each amino acid
-recognized amino acids by size, charge, and R group
-recognizes tRNA by specific sequences at specific spots
-need ATP
-forms charge tRNA

siRNA

-small inference RNA
-fragments of dsRNA cleaved by Dicer
-combine with protein to form RISC
-RISC bind to mRNA and silence the gene by degradation
-for bind to happen fragment from either strand must be highly homologous with mRNA sequence

RNAi

-limit invasion of foreign genes, and censor their own gene expression
-has two classes of snRNA (siRNA and miRNA)
-triggered by dsRNA which is chopped by Dicer
-this produces snRNA
-snRNA bind to mRNA and silence its translation

RISC

-RNA induced silencing complex
-snRNA + protein
-bind to mRNA for gene silencing

Why does DNA contain thymine

-cytosine naturally deaminates to form uracil
-cell would not have any way of knowing whats a mutated C and a regular U
-thymine is a (5-methyl-U) to solve this problem, if cythosine becomes U then repair enzyme knows DNA has no U.

Cytosine deaminated into uracil

Nucleic acid hydrolysis

-break bond in nucleotide backbone
-important to manipulate polymer molecules

DNA hydrolysis

-resistant to dilute base
-depurinated in dilute acid
-with 1mM/HCl purine glycosidic bond is broken, giving a apurinic acid
-pyrimidine is not affected, therefore neither is the backbone structure

RNA hydrolysis

-resistant to dilute acid
-hydrolyzed by diluted base (NaOH)
-gives 2' or 3' phosphate ester

RNA hydrolysis steps

1- OH from diluted base abstract 2' H, leaving a O anion
2-the new anion is nucleiophilic, and attract P from phosphate group
3- 5' phosphate ester bond is broken
4- 2' 3' cyclic phosphodiester is formed, but its unstable
5- 2' or 3' phosphate ester are formed randomly

Nuclease

-nuclease enzyme that hydrolyzes nucleic acid
-found in some digestive organs like pancreas
-fungi and snake venom is a good source of nuclease
-phosphodiesterase

Nuclease Phosphodiesterase

-cleave can occur on "a" or "b" position
-"a" usually on 3' end
- "b" on 5' termini
-can also cleave endo or exo

where does exo a phosphodiesterase cleaves?

Cleavage on the a side (3' end), leaves the phosphate attached to the 5'-position of the adjacent nucleotide

where does exo b phosphodiesterase cleaves?

-5' termini
-b-side hydrolysis yields 3'-phosphate products

Nuclease specificity

-DNase or RNase
-ds or ss
-base
-nucleotide sequence (4-8 bp)
-nonspecific = nuclease

Exonuclease

degrade nucleic acids by sequentially removing nucleotides from their ends

snake venom phosphodiesterase

-Exonuclease
-acts by a cleavage and starts at the free 3'-OH end of a
polynucleotide chain, liberating nucleoside 5'-monophosphates.

bovine spleen phosphodiesterase

-Exonuclease
-enzyme starts at the 5'-end of a nucleic acid, cleaving b and releasing 3'-NMPs

Restriction endonuclease

-found in bacteria
-attach foreign bodies by cleaving dsDNA
-degrade DNA by chopping it into noninfective fragments
-classified into Type I, II and III

Which type of restriction enzyme need ATP?

Type I and III

What unique about Type I enzyme?

it cleaves randomly

Besides hydrolyze DNA, what else can Type I and III do?

catalyze chemical modification of DNA through addition of methyl groups to specific bases

Type II restriction enzyme

-widespread application in the cloning and sequencing of DNA molecules
-not ATP dependent
-they do not modify DNA by methylation or other means
-they cut DNA within or near particular nucleotide sequences that they specifically recognize
-usually 4-6 bp twofold axis of symmetry
-EcoRI
:sticky" or "blunt" terminal

Twofold axis of symmetry

EcoRI

-sequence GAATTC
-when it find sequence it causes a staggered, double-stranded break by hydrolyzing each chain between the G and A residues
-staggered cleavage leaves strand with 5' sticky ends
-there can be recombination
-first restriction enzyme found in the R strain of E. coli

Sticky ends

"sticky" ends can be joined together to create new combinations of DNA sequence. If the fragments are derived from DNA molecules of different origin, novel recombinant forms of DNA are created.

Six-cutter

-enzymes such as EcoRI or BamHI, will find their unique hexanucleotide sequences on the average once in every 4096 (4^6)bp
-the fragments generated by it are approximately the size of prokaryotic genes. This property makes these enzymes useful in the construction and cloning of genetically useful recombinant DNA molecules.

Naming Restriction Enzyme

-3 letter italicized cond
1st letter = genus
2nd and 3rd = species
following letter denotes strain

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