ch 8 nucleotides and nucleic acids

Nucleotides- role in cellular metabolism

•have a variety of roles in cellular metabolism:

–energy currency in metabolic transactions

–essential chemical links in the response of cells to hormones and other extracellular stimuli

–structural components of an array of enzyme cofactors and metabolic intermediates

constituents of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)

Gene

•segment of a DNA molecule that contains the information required for the synthesis of a functional biological product, whether protein or RNA

Functions of DNA

–storage of biological information

transmission of that information to the next generation

The functions of RNA

•several classes of RNA:

–ribosomal RNAs (rRNAs) = components of ribosomes

–messenger RNAs (mRNAs) = intermediates in protein synthesis

–transfer RNAs (tRNAs) = adapter molecules that translate the information in mRNA into a specific amino acid sequence

noncoding RNAs (ncRNAs) = wide variety of functions

Components of nucleotides

have three components:

a nitrogenous base (pyrimidine or purine)

a pentose

1+ phosphates

Nucleoside

The molecule without a phophate group

Nucleotide bonds

N-β-glycosyl bond = covalently joins the 1′ carbon of the pentose to the base (at N-1 of pyrimidines and N-9 of purines)

formed by removal of the elements of water

the phosphate is esterified to the 5′ carbon

Nitrogenous bases

major purine bases:

adenine (A) = in DNA and RNA

guanine (G) = in DNA and RNA

major pyrimidine bases:

cytosine (C) = in DNA and RNA

thymine (T) = only in DNA

uracil (U) = only in RNA

Nucleotide nulcleic acid Nomenclature

Nucleotide pentose

two kinds of pentoses:

2′-deoxy-d-ribose = in DNA

D-ribose = in RNA

both are in their β-furanose (closed five-membered ring) form

Deoxyribonucleotides

structural units of DNA

also called deoxyribonucleoside 5′-monophosphates, deoxynucleotides, and deoxynucleoside triphosphates

Ribonucleotides

structural units of RNA

also called ribonucleoside 5′-monophosphates

Minor purine and pyrimidine bases

Methylated forms most common in DNA

Phosphodiester linkage

covalent bond that joins successive nucleotides of both DNA and RNA

between the 5′-phosphate group of one nucleotide unit and the 3′-hydroxyl group of the next nucleotide

Hydorlysis of DNA and RNA

under alkaline conditions:

RNA is rapidly hydrolyzed due to the presence of 2′-hydroxyl groups

DNA is not rapidly hydrolyzed

Oligonucleotide

Short (typically <50 nucleotides) nucleic acid

Polynucleotide

Longer nucleic acid

What are the properties if nucleotide bases that affect eh 3 dimensional stucture of nucleic acids?

weakly basic compounds

aromatic molecules

because most bonds in the ring have partial double-bond character:

pyrimidines are planar

purines have a slight pucker

 

What are the pryimidine andpurine bases that may exist as tautomers forms called?

may exist in readily interconverted forms called tautomers

lactam

lactim

double lactim

Absorption spectra of common nucleotides

all nucleotide bases absorb UV light

strong absorption near 260 nm

Solubility of nucleotides

hydrophobic and relatively insoluble in pH 7.0 water

leads to stacking interactions (van der Waals and dipole-dipole)

charged and more soluble at acidic or alkaline pH values

Base pairs

hydrogen-bonding patterns between complementary strands of nucleic acids

A bonds specifically to T (or U)

G bonds specifically to C   

Hierchial levels of nucleic acid structure

primary structure = covalent structure and nucleotide sequence

secondary structure =  regular, stable structure taken up by some or all the nucleotides

tertiary structure = complex folding of large chromosomes or the elaborate folding of tRNA or rRNA structures

Watson crick model for the structure of DNA

offset pairing of the two strands creates a major groove and a minor groove

3 hydrogen bonds form between G and C

2 hydrogen bonds form between A and T

parallel

3′ ,5′ -phosphodiester bonds run in the same direction

DNA strands are antiparallel

3′ ,5′ -phosphodiester bonds run in opposite directions

ultimately confirmed by x-ray analysis

Double helix measurements

10.5 base pairs

36 A (3.6nm0

Complementary

double-helical DNA strands are complementary:

when A occurs in one chain, T is found in the other

when G occurs in one chain, C is found in the other

hydrogen bonding does not contribute significantly to stability of the structure

How is the double helix stabilized

metal cations that shield the negative charges of backbone phosphates

base stacking interactions between successive base pairs

successive G≡C or C≡G are stronger than successive A=T or T=A

duplexes with higher G≡C context are more stable

Replication of DNA

Step 1: preexisting or “parent” strands become separated

Step 2: each “parent” strand serves as a template for the biosynthesis of a complementary “daughter strand”

What does structural variation in DNA refelects

different possible conformations of the deoxyribose

rotation about the contiguous bonds making up the phosphodeoxyribose backbone

free rotation about the C-1′–N-glycosyl bond

B-form DNA

the Watson-Crick structure

most stable for a random-sequence DNA molecule under physiological condition

A-form DNA

right-handed double helix with a wider helix, 11 bp/turn, and a tilted plane

favored in solutions devoid of water

Z- form DNA

left-handed helix with 12 bp/turn and a backbone with a zig-zag appearance

appears more slender and elongated

Palindrome

region of DNA that is identical when read either forward or backward

applied to regions of DNA with inverted repeats

Mirror repeat

Sequence when the inverted repeat occurs within each individual strand

Hairpin and cruciform structures

Form form the self complementarity within each strand

Tetraplex DNA

occur when four DNA strands pair

occur readily only for DNA sequences with a very high proportion of G residues

G tetraplex = very stable

Messenger RNA (mRNA)

Portion of cellular RNA carrying gentic information from DNA tot he ribosome

Transcription

Process by which mRNAs are formed on DNA template

Monocistronic

Codes for only one polypeptide

-most mRNAs in eukaryotes

Polycistronic

Codes for 2 + different polypeptides

-occurs in bacteria and archaea

MRNa structure

mRNA is always single-stranded

right-handed helical conformation

dominated by base-stacking interactions

strongest between two purines

can base pair with complementary regions of DNA or RNA

paired strands are antiparallel

Secondary suture of RNAs

structure of complementary RNA strands is an A-form right-handed double helix

breaks caused by mismatched or unmatched bases result in bulges or internal loops

 

internal loops form between palindromic sequences

Denaturation or melting of the double helix

due to pH extremes or high temperatures

disrupts hydrogen bonds and base-stacking interactions

Anneal

Process by which two stands spontaneously rewind when temperature or pH is returned to its normal range

-two step process

Hypochromic effect

The observed decrease int eh absorption of UV light when complementary strands are paired

Hyperchromic effect

The observed increase in the absorption of UV light when a double stranded nucleic acid is denatured

-monitoring UV absorption at 260nm can detect the transition from the double stranded to the single stranded DNA

Denaturation temperature tm

temperature at which ½ of DNA is present as separated single strands

increases with content of G≡C base pairs

Partially denatured DNA

Denatured regions form bubbles

Often rich in A=T base pairs

Mutations

Alteration sin DNA structure that produce permanent changes in genetic information encoded

-linked to aging and carcinogenesis

Delamination reactions

deamination = spontaneous loss of exocyclic amino groups

deamination of cytosine to uracil = ~100 events/day

recognized as foreign in DNA and removed

almost certainly why DNA contains thymine rather than uracil

Depurination reactions

hydrolysis of the N-β-glycosyl bond between the base and the pentose

creates an AP (apurinic, apyrimidinic) site or abasic site

more common with purines

Reactions promoted by radiation

UV light causes:

cyclobutane pyrimidine dimers

6-4 photoproduct

ionizing radiation (x-rays and gamma rays) causes:

ring opening

base fragmentation

breaks in the covalent backbone of nucleic acids

What are reactive chemicals that damage DNA?

nitrous acid precursors = deaminating agents

alkylating agents = generate modified nucleotides nonenzymatically

How does alkylating agents can alter certain bases of DNA

can methylate guanine to O6-methyl-guanine, which cannot base-pair with cytosine

DNA damage by oxidative damage

reactive oxygen species (hydrogen peroxide, hydroxyl radicals, superoxide radical) damage DNA

hydroxyl radicals are responsible for most oxidative DNA damage

cells have an elaborate defense system to destroy reactive oxygen species 

Polymerase chain reaction (PCR)

method of amplifying DNA segments of interest

relies on DNA polymerases (enzymes that synthesize DNA from deoxyribonucleotides (dNTPs) using a DNA template)

DNA polymerases add nucleotides to the 3′ ends of preexisting strands called primers

Pc technology id highly sensitive

PCR can detect and amplify just one DNA molecule in almost any sample type

Uses of PCR:

cloning of rare, undegraded DNA segments from 40,000+ years ago

tracing evolution

potent tool in forensic medicine

detecting viral infections and cancers before they cause symptoms

prenatal diagnosis of genetic diseases

Sanger sequencing

dideoxy chain-termination sequencing

nucleotide analogs called dideoxynucleoside triphosphates (ddNTPs) interrupt synthesis

Reversible terminator sequencing

developed by Illumina

uses four different modified deoxynucleotides (A, T, G, and C), each with a particular fluorescent label and a 3′ blocking group

SMRT sequencing

technology allowing read lengths averages up to 30,000-40,000 bp

lower throughput, higher cost, and higher error rate than the Illumina approach 

Sequencing depth

Contigs

the number of times that a particular nucleotide in a genome is sequenced, on average

Contigs- long contiguous sequences that are assembled form overlaps