biochemistry nucleic acids - … · nucleic acids (nas) • cells within a living organism are able...

BIOCHEMISTRY

Nucleic Acids

BIOB111

CHEMISTRY & BIOCHEMISTRY

Session 13

Session Plan

• Types of Nucleic Acids

• Nucleosides

• Nucleotides

• Primary Structure of Nucleic Acids

• DNA Double Helix

• DNA Replication

• Types of RNA

Stoker 2014, Figure 23-2 p843

Nucleic Acids (NAs)• Cells within a living organism are able to produce exact replicas of themselves

– Cells contain all the information needed to produce the complete organism• Information stored in nucleic acids

• Nucleic acids are found in the nucleus & are acidic– Nucleic acids are polymers of Nucleotides (the monomers)

• DNA – Deoxyribonucleic acid– Found in the nucleus, stores & transfers genetic info.

– Passed from one cell to another new cell during cell division

• RNA – Ribonucleic acid– Found in the nucleus & cytoplasm

– Primary function is to synthesize proteins

• The hereditary information, required for protein synthesis– Stored in the molecules of DNA in the nucleus & mitochondria (mitochondrial DNA)

Nucleus

Cell

rRNA tRNA

DNA

mRNA

mRNA

Protein

Chromosomes• The DNA in the nucleus wraps

around proteins called Histones– Histones join together into a fibre,

forming Chromosomes

• Chromosome mass is about 15% DNA & 85% proteins

• Cells of different organisms have different numbers of chromosomes in cell nuclei– The nucleus of human somatic cells

contains 23 pairs of chromosomes (autosomes)

• Includes the Two sex chromosomes (XX or XY)

Tortora & Grabowski 2003, Figure 3.26, p.85

Nucleotides

• Nucleic acids (DNA & RNA) are polymers of Nucleotides

• Nucleotides are the monomer building blocks of nucleic acids– Just like monosaccharides are building blocks of polysaccharides &

amino acids are building blocks of proteins.

• A Nucleotide has 3 components:– Nitrogen-Containing Base

– Pentose Monosaccharide

– Phosphate Group

N-containing Bases• 3 derivatives of Pyrimidine (small):

– Cytosine ( C ) – DNA & RNA

– Thymine ( T ) – DNA only

– Uracil ( U ) – RNA only

• 2 derivatives of Purine (large): – Adenine ( A )

– Guanine (G )

• Both purines and pyrimidines

are present in DNA & RNA

Pentose Sugars

• Pentose monosaccharides – Ribose – in RNA

– Deoxyribose – in DNA. • Deoxyribose is lacking O atom on C2’

Lacks Oxygen in position 2

Phosphate Group

• The phosphate group is derived

from phosphoric acid (H3PO4)

– The cellular pH leads to full dissociation of the phosphoric acid, producing a hydrogen phosphate ion (HPO4

2-).

• Common abbreviations Pi = Inorganic Phosphate

Nucleoside

• Base + Sugar = NUCLEOSIDE

Formation of a glycosidic bond

Nucleotide

• Nucleoside + Pi attached to C5’ position of pentose sugar via phosphate-ester bond

• Base + Sugar + Phosphate group = NUCLEOTIDE

Stoker 2014, Table 22-1 p792

Structure of Nucleic Acids• Nucleic acids have primary & secondary structure (like proteins)

– Nucleic acids = polymers of Nucleotides

• Primary structure of both DNA & RNA = the sequence of nucleotides

• The primary structure has 2 parts:– 1) Backbone composed of sugars & phosphate groups

• Constant through entire molecule

– 2) Nitrogen Bases as side chains

• Sequence of bases is variable & distinguishes 1 nucleic acid from another


Primary Structure of Nucleic Acids• Primary structure of a nucleic acid:

The Sequence of Bases attached to the sugar units of the backbone changes

• Each non-terminal Pi (not at the ends) of the backbone is bonded

to 2 sugar molecules via a 3’,5’-phosphodiester bond

– One phosphoester bond between

phosphate and C5’ of one sugar

– Another phosphoester bond between

phosphate and C3’ of the next sugar

• The nucleotide chain has a direction– 5’ end: Carries a free Pi attached to C5’ of the sugar

• 3’ end: has a free –OH on C3’ of the sugar

• By convention, the sequence of bases on

a nucleic acid strand (DNA or RNA) is read from the

5’ end to the 3’ end


Secondary Structure of DNA

• The DNA of every animal & plant has a unique base composition

– The relationships between the bases is always the same

• % A = % T

• % C = % G

• A/T and C/G form hydrogen bonds to hold together the double stranded DNA helix

– In 1953, Watson & Crick received the Nobel Price for ‘explaining’ the secondary structure of DNA as a Double Helix

– Human DNA contains 30% each of A & T & 20% each of C & G


DNA Double Helix

• Double Helix = 2 strands of DNA in their primary

structure

– Wound up around each other like a spiral staircase

• The sugar + Pi backbones of the 2 strands are like

banisters of the staircase

• The bases of both strands extend inwards towards the

bases of the other strand & are bonded together via

Hydrogen bonds

• The 2 strands are anti-parallel,

run in opposite directions

– One strand runs 5’→3’ & the other 3’→5’ directionStoker 2014, Figure 22-7 p798

Base Pairing

• Base Pairing:– Always 1 purine base (large) & 1 pyrimidine base (small)

linked together via Hydrogen bonds

– The interior of the Double helix is small

• 2 large purine bases would not fit

• 2 small pyrimidine bases would be too far apart to form Hydrogen bonds

• When linking together via Hydrogen bonds, the bases form the Secondary structure of DNA & are known as Complementary bases– Two bases that form hydrogen bonds together are complementary base pairs

• A/T, C/G are complementary base pairs

Complementary Base Pairs

In DNA

• A T

• G C

In RNA

• A U

• G C



HydrogenBonding is more

favourable between

A = TG ≡ C

DNA Double Helix

• The Double Helix of DNA = 2 complementary DNA strands.

– If the sequence of bases in 1 strand is known, the sequence of

bases in the complementary strand can be predicted

– 5’ A – A – T – G – C – A – G – C – T 3’

– 3’ T – T – A – C – G – T – C – G – A 5’

Known strand

Unknown strand

Which part of the nucleotide is considered

the backbone and which is the side chain?

Which part of the nucleic acid molecule

(collection of nucleotides e.g. DNA, RNA)

is responsible for forming the 2ᵒ structure (helical spiral)?

What type of intermolecular force is responsible for forming

the 2ᵒ structure within nucleic acids?

Which bases form these forces together? Why?

Key concept: structure of nucleotide, nucleic acid

Attempt Socrative questions: 1 to 3

Google Socrative and go to the student login

Room name:

City name followed by 1 or 2 (e.g. PERTH1)

1 for 1st session of the week and 2 for 2nd session of the week

DNA Replication

• DNA replication: The process by which a DNA molecule

produces an exact duplicate of itself

– Takes place in the nucleus when the parent DNA divides into 2

daughter DNA molecules

– Each daughter DNA molecules is identical to the parent DNA molecule

• Replication uses the same principle of base pairing as

encountered in the Secondary structure of DNA double helix

DNA Replication• Both of the DNA strands (two) forming the double helix serve as

Templates for copying

• The enzyme DNA-helicase unwinds the double helix & breaks the hydrogen bonds between the bases– Like undoing a zipper

– Each DNA strand without the double stranded DNA helix is separated

– The 2 separated strands each act as a template for the synthesis of a new complementary strands

• Replication fork = the point at which the double helix is unwinding– Constantly moving as the position of the fork shifts

DNA Replication

• After the DNA helix has unwound:– The bases of the separated strands are not connected by hydrogen bonds anymore

– The DNA template strands can now pair with free individual nucleotides present in the nucleus (C≡G & A=T) one at a time

• As the new bases are added in they form new hydrogen bonds with the old strand (the template)

– End result of DNA replication is two double stranded DNA helices

• DNA-polymerase enzyme checks if the pairing of bases

(between template and new strand) is correct– Also joins the new bases to a new backbone

– Catalyzes formation of new phosphodiester bonds between nucleotides• Connects the phosphate of one nucleotide and the sugar of the second nucleotide


DNA Replication

DNA Replication

• Each of the 2 daughter molecules of double-stranded DNA, formed during replication:– 1 strand from the original parent molecule

– 1 newly formed strand

• The 2 daughter DNA molecules are synthesized in different ways

– Leading strand: Continuously grows in 5’→3’ direction

• DNA-polymerase can function only in 5’→3’ direction.

– Lagging strand: Synthesized in short segments = Okazaki Fragments

• Okazaki fragments are sequences of about 200 nucleotides made in 5’→3’ direction

– Okazaki fragments are eventually joined together by the enzyme DNA-ligase

DNA Replication

• Replication usually occurs at multiple sites within the DNA molecule & proceeds in both directions.– This multiple-site replication enables rapid DNA synthesis.

Note:

• Anti-metabolites: drug used in chemotherapy– Anti-cancer drugs that interfere with DNA-replication in cancer cells, causing

them to die• Cannot produce more cancer cells

• Examples: 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), Floxuridine


Parent strand 1Daughter strand 1

Stoker 2104, p804

Summary of DNA Replication

What is the purpose of replicating DNA?

After one round of DNA replication there are

two double stranded DNA helices.

What types of strands do the double stranded DNA

helices contain, new strands, template strands or both?

Why is it that an existing DNA strand can be is used

as a template to create a DNA new strand, which is

complementary to the original strand?

Key concept: DNA replication

Attempt Socrative questions: 4 and 5


Room name:



Differences between DNA & RNA

DNA RNA

Pentose Sugar

in backbone

Deoxyribose Ribose

Complementary

Base Pairs

A = T

C ≡ G

A = U

C ≡ G

Strand Double helix Single strand

Size Very large

(1-100 million nucleotides)

Much smaller

(75-few 1,000 nucleotides)

RNA Molecule

• Even though the RNA molecule is a single strand it often folds

upon itself in certain parts and forms double-helical regions


Secondary Structure of RNA

• RNA forms only a single strand, which is the Primary structure

– Sequence of bases

• Secondary structure

– Portions of the strand fold onto itself, forming loops of double helical regions

• RNA contains Uracil instead of Thymine


Types of RNA Molecules

• There are 5 types of RNA but only 3 types will be

discussed:

– Messenger RNA

– Ribosomal RNA

– Transfer RNA

Messenger RNA – mRNA

• mRNA carries the genetic information from the DNA in the nucleus

to the site of protein synthesis in the cytoplasm

• The mRNAs nucleotide sequence is complementary to one of the DNA strands

– Therefore: DNA sequence dictates the order that the amino acids will be fused to form the protein

• mRNA is not very stable, it is synthesized when needed & then degraded– Size of the mRNA varies according to the length of the protein to be synthesized

• The average size is about 750 nucleotides

• Constitutes about 5-10% of overall RNA mass

Ribosomal RNA – rRNA

• Combines with specific proteins & forms ribosomes

– Ribosomes are the site of protein synthesis

• The composition of ribosomes:

– Proteins – 35%

– rRNA – 65%

• rRNA molecules are large

– The most abundant RNA-type, constitutes about 75-80% of overall RNA mass

Transfer RNA – tRNA • Transports amino acids to the

site of protein synthesis (= ribosomes)

– Contain 75-90 nucleotides

– Are the smallest of the RNAs

• Anticodon is complementary to a particular mRNA sequence

• There is at least one different tRNA molecule for each of the 20 standard amino acids

– Some amino acids have more than one tRNA


2D structure

3D structure

Transcription – RNA Synthesis

• Transcription is the process by which DNA directs the synthesis of mRNA– rRNA & tRNA are produced in the same way– Takes place in the nucleus

• A nucleotide sequence on DNA is copied / transcribed onto a RNA (m, r or t) molecule– The information for the protein synthesis is in DNA (nucleus) but the amino

acids are in the cytosol• Information to make protein must get out of the nucleus >>> does so via mRNA

• The info is copied from DNA to RNA in a slightly different format

– DNA: A, C, G, T

– RNA: A, C, G, U

Transcription Steps

• 1) The enzyme RNA-polymerase unwinds a portion of DNA double helix exposing one particular gene– Gene = a DNA segment coding for a specific mRNA / protein

– Only one strand of the DNA molecule is transcribed

• 2) On the exposed DNA strand there is always:– Initiation signal (start) at the beginning of the gene

– Termination sequence (stop) at the end of the gene

• Recognized by the RNA-polymerase (moves along DNA in 3’ → 5’ direction) as “START” & “STOP”

Transcription Steps

• 3) Transcription ends when RNA-polymerase reads “STOP”

– The newly formed mRNA molecule (synthesized in the 5’ → 3’ direction) as well as the RNA-polymerase are released

– DNA rewinds to re-form the original double helix

– tRNA and rRNA are synthesised in the same way

• After the RNA molecules have been synthesised:– RNA moves out of the nucleus through

the nuclear membrane pores into the cytoplasm


Splicing

Protein Synthesis Overview

• The genetic information contained within the DNA is expressed in the cell function through protein synthesis– Protein synthesis is under the direction of DNA

• The whole process occurs in 2 phases:– TRANSCRIPTION = RNA Synthesis (in the nucleus)

– TRANSLATION = Protein Synthesis (in the cytoplasm)

In the nucleus In the cytoplasm

The Genetic Code• The genetic code is universal

– The same codon specifies the same amino acid in a cell of all living organisms

• Initiation (start) & termination (stop) codons exist:

– AUG = always “START” codon & codes for Met

– UAA, UAG & UGA = “STOP” codons & do not code for any AA.

• The genetic code is highly degenerate – Some amino acids are coded for by more than 1 codon

• Arg, Leu & Ser have 6 codons (synonyms)

• Other AAs have 2 or more codons

• Met & Trp have only single codon

tRNA & Anticodons

• The Anticodon of tRNA molecule complementary aligns with the codon of mRNA forming protein

– tRNA delivers a specific amino acid to grow the protein chain

Stoker 2014,

Figure 22-21 p820

Summary

• DNA

• mRNA

• Protein

Mutation• Mutation:

Error in base sequence of a gene, that alters the DNA structure– Any errors in the DNA structure are reproduced during replication of DNA

• Passed on during transcription onto mRNA

• Altered genetic information can cause changes in the amino acid sequence of a protein

– Altered function of the protein >>> potentially lethal consequences

• When a protein is not functioning correctly due to hereditary mutation in reproductive cells

– Genetic disease: phenylketonuria, albinism, cystic fibrosis,

sickle cell anaemia, haemophilia

• Cancer can result when:– Person is born with a correctly functioning protein in somatic cells & it becomes

dysfunctional due to mutation

Mutations• Substitution Mutation

– A correct base in DNA is substituted by an incorrect one

• Leading to a change in one mRNA codon & the incorrect AA is incorporated into the protein

• Frame Shift Mutation– A base is added or deleted from the

correct DNA sequence• Shifting the sequence frame of the

DNA

• Leading to a shift in the mRNA codon sequence

– All AA’s downstream of the frame shift site will be altered

Timberlake 2014, Figure 19, p.792

Attempt Socrative questions: 6 to 10


Room name:



Mutagens • Mutagen is a chemical or other agent that causes mutation

– Ionizing radiation (X-rays, UV, γ-rays)

– Nitrous acid (HNO2) converts C to U (codon can change from CGG to UGG)

• Possibly damaging DNA

• Nitrates, nitrites (preservatives in foods, e.g. hot dogs) & nitrosamines can form nitrous acid in the body.

• Carcinogen = a mutagen that can cause cancer

• The body has repair enzymes that can correct some mutations– Function by recognizing & replacing altered bases

– However sometimes the damage is not repaired & the mutation persists

MutagensViruses

• The virus attaches to the cell to

transfer its genetic material into the cell– Alters the original gene, causing mutation

• E.g. Simian vacuolating virus 40 (SV40 virus)

• Human Papilloma virus

• Genetic material of virus:– DNA virus: Hepatitis B virus, Human

herpes virus

– RNA virus: Hepatitis C virus

Asbestos

• Exposure to asbestos mutates p53 gene– Alters tumour suppressing role of the

gene• Cause lung cancer

• Once mutated P53 gene no longer suppresses tumours

Pesticides

• Handling or inhaling pesticides causes gene mutation

– Growth abnormalities were observed in children born to pregnant women exposed to the pesticide ‘Endosulfan’

• Many pesticides were banned including Endosulfan & DDT

Smoking & Alcohol Consumption

• Common mutagens when consumed in excess

– Many of the chemicals in cigarettes are mutagens

• Benzene, polonium-210, benzo(a)pyrene and nitrosamines

– Mutation caused by chain smoking is similar to that of the exposure to asbestos

– Consumption of excess alcohol causes sperm cell mutation contributing to genetic defects in the offspring

Recombinant DNA, Genetic Engineering & Diagnosis

Recombinant DNA

• DNA molecules that contain genetic material from 2 different

organisms

– Produced by splicing a desired gene from one organism to the DNA of

another organism

Genetic Engineering (Biotechnology)

• A process in which an organism is intentionally changed at the

molecular (DNA) level,

so that it exhibits different traits

• First genetically engineered organism were bacteria (1973) &

mice (1974)

• Insulin-producing bacteria were commercialized in 1982

• Genetically modified food crops have been available since 1994

Polymerase Chain reaction

• PCR is an excellent

technique for the rapid

detection of pathogens,

including those difficult to

culture• Virology

• Mycology

• Parasitology

• Microbiology

Polymerase Chain Reaction (PCR)

• PCR is a method for rapidly producing multiple copies of a DNA nucleotide sequence (gene)– Utilises the DNA-polymerase enzyme

– Allows to produce billions of copies of a specific gene in a few hours

• DNA that is available in very small quantities can be amplified to quantities large enough to analyze

• Used for disease diagnosis:– Genetic disorders like cystic fibrosis

– Detecting pathogens (HIV) in the body

– Forensics: DNA fingerprinting

Stoker 2014, Table 22-3 p835

Readings & Resources• Stoker, HS 2014, General, Organic and Biological Chemistry, 7th edn,

Brooks/Cole, Cengage Learning, Belmont, CA.

• Stoker, HS 2004, General, Organic and Biological Chemistry, 3rd edn, Houghton Mifflin, Boston, MA.

• Timberlake, KC 2014, General, organic, and biological chemistry: structures of life, 4th edn, Pearson, Boston, MA.

• Alberts, B, Johnson, A, Lewis, J, Raff, M, Roberts, K & Walter P 2008, Molecular biology of the cell, 5th edn, Garland Science, New York.

• Berg, JM, Tymoczko, JL & Stryer, L 2012, Biochemistry, 7th edn, W.H. Freeman, New York.

• Dominiczak, MH 2007, Flesh and bones of metabolism, Elsevier Mosby, Edinburgh.

• Tortora, GJ & Derrickson, B 2014, Principles of Anatomy and Physiology, 14th edn, John Wiley & Sons, Hoboken, NJ.

• Tortora, GJ & Grabowski, SR 2003, Principles of Anatomy and Physiology, 10th edn, John Wiley & Sons, New York, NY.

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