biochem nucleic acids
DESCRIPTION
Nucleic AcidsTRANSCRIPT
NUCLEIC ACIDS
Topic Outline:
History of Nucleic Acids Structure and Function Types of Nucleic Acids
1. DNA2. RNA
Central Dogma of Life
Friedrich Miescher in 1869
isolated what he called nuclein from the nuclei of pus cells
Richard Altmann in 1889
Nuclein was shown to have acidic properties, hence it became called nucleic acid
1920s
the tetranucleotide hypothesis was introduced
The Tetranucleotide hypothesis
Up to 1940 researchers were convinced that hydrolysis of nucleic acids yielded the four bases in equal amounts.
Nucleic acid was postulated to contain one of each of the four nucleotides, the tetranucleotide hypothesis.
Takahashi (1932) proposed a structure of nucleotide bases connected by phosphodiester linkages.
The Tetranucleotide hypothesis
adenine uracil
cytosine guaninephosphate
phosphate phosphate
phosphate
pentose
pentosepentose
pentose
Astbury and Bell in 1938
First X-ray diffraction pattern of DNA is published.
The pattern indicates a helical structure, indicated periodicity.
X-ray diffraction of DNA
Wilkins & Franklin (1952): X-ray crystallography
Avery, MacLeod, and Mc Carty in 1944
demonstrate DNA could “transform” cells.
Supporters of the tetranucleotide hypothesis did not believe nucleic acid was variable enough to be a molecule of heredity and store genetic information.
DNA is Genetic Material
Erwin Chargaff in late 1940s
used paper chromatography forseparation of DNA
hydrolysates. Amount of adenine is equal to
amount of thymine and amount of guanine is equal to amount of cytosine.
Hershey and Chase in 1952
confirm DNA is a molecule of heredity.
The Hershey-Chase Experiment
The Hershey-Chase Experiment
Watson and Crick in 1953
determine the structure of DNA
Watson & Crick Base pairing
Francis Crick in 1958
proposes the “central dogma of molecular biology” .
Kornberg purifies DNA polymerase I
1969
Entire genetic code determined
Nucleic Acids
• Nucleic Acids are very long, thread-like polymers, made up of a linear array of monomers called nucleotides.
• Nucleic acids vary in size in nature
• tRNA molecules contain as few as 80 nucleotides
• Eukaryotic chromosomes contain as many as 100,000,000 nucleotides.
Two types of nucleic acid are found
Deoxyribonucleic acid (DNA) Ribonucleic acid (RNA)
DNA and RNA
DNAdeoxyribonucleic acidnucleic acid that stores genetic informationfound in the nucleus of a mammalian cell.
RNAribonucleic acid3 types of RNA in a cellRibosomal RNAs (rRNA) are components of ribosomesMessenger RNAs (mRNA) carry genetic informationTransfer RNAs (tRNA) are adapter molecules in translation
The distribution of nucleic acids in the eukaryotic cell DNA is found in the nucleus
with small amounts in mitochondria and chloroplasts
RNA is found throughout the cell
The nucleus contains the cell’s DNA (genome)
Nucleus
RNA is synthesized in the nucleus andexported to the cytoplasm
Nucleus
Cytoplasm
DNA as genetic material: The circumstantial evidence1. Present in all cells and virtually restricted to the
nucleus
2. The amount of DNA in somatic cells (body cells) of any given species is constant (like the number of chromosomes)
3. The DNA content of gametes (sex cells) is half that of somatic cells. In cases of polyploidy (multiple sets of chromosomes) the DNA content increases by a proportional factor
4. The mutagenic effect of UV light peaks at 253.7nm. The peak for the absorption of UV light by DNA
NUCLEIC ACID STRUCTURE
Nucleic acids are polynucleotides
Their building blocks are nucleotides
NUCLEOTIDE STRUCTURE
PHOSPATE SUGAR
Ribose or Deoxyribose
NUCLEOTIDE
BASEPURINES PYRIMIDINES
Adenine (A)Guanine(G)
Cytocine (C)Thymine (T)Uracil (U)
All nucleotides contain three components:1. A nitrogen heterocyclic base2. A pentose sugar3. A phosphate residue
Nucleotide Structure
Ribose is a pentose
C1
C5
C4
C3 C2
O
RIBOSE DEOXYRIBOSE
CH2OH
H
OH
C
C
OH OH
C
O
H HH
C
CH2OH
H
OH
C
C
OH H
C
O
H HH
C
Spot the difference
Ribonucleotides have a 2’-OHDeoxyribonucleotides have a 2’-H
Chemical Structure of DNA vs RNA
THE SUGAR-PHOSPHATE BACKBONE
The nucleotides are all orientated in the same direction
The phosphate group joins the 3rd Carbon of one sugar to the 5th Carbon of the next in line.
P
P
P
P
P
P
ADDING IN THE BASES
The bases are attached to the 1st Carbon
Their order is important It determines the genetic information of the molecule
P
P
P
P
P
P
G
C
C
A
T
T
DNA IS MADE OF TWO STRANDS OF POLYNUCLEOTIDE
P
P
P
P
P
P
C
G
G
T
A
A
P
P
P
P
P
P
G
C
C
A
T
T
Hydrogen bonds
DNA IS MADE OF TWO STRANDS OF POLYNUCLEOTIDE
The sister strands of the DNA molecule run in opposite directions (antiparallel)
They are joined by the bases Each base is paired with a specific partner:
A is always paired with T G is always paired with C“Purine with Pyrimidine”
The sister strands are complementary but not identical
The bases are joined by hydrogen bonds, individually weak but collectively strong
There are 10 base pairs per turn
Structure of Nucleotide Bases
Purines & Pyrimidines
5’ End
3’ End
Nucleotides arelinked byphosphodiesterbonds
From DNA to Protein
DNA to Protein
DNA acts as a “manager” in the process of making proteins
DNA is the template or starting sequence that is copied into RNA that is then used to make the protein
Central Dogma
One gene – one protein
Central Dogma
This is the same for bacteria to humans
DNA is the genetic instruction or gene DNA RNA is called TranscriptionTranscription
RNA chain is called a transcripttranscript RNA Protein is called TranslationTranslation
Expression of Genes
Some genes are transcribed in large quantities because we need large amount of this protein
Some genes are transcribed in small quantities because we need only a small amount of this protein
Nucleotides as Language We must start to think of the
nucleotides – A, G, C and T as part of a special language – the language of genes that we will see translated to the language of amino acids in proteins
Genes as Information Transfer
A genegene is the sequence of nucleotides within a portion of DNA that codes for a peptide or a functional RNA
Sum of all genes = genomegenome
STEP 1 – DNA REPLICATION
DNA Replication
SemiconservativeSemiconservative Daughter DNA is a
double helix with 1 parent strand and 1 new strand
Found that 1 strand serves as the template for new strand
DNA Template
Each strand of the parent DNA is used as a templatetemplate to make the new daughter strand
DNA replication makes 2 new complete double helices each with 1 old and 1 new strand
Replication Origin
Site where replication begins 1 in E. coli 1,000s in human
Strands are separated to allow replication machinery contact with the DNA Many A-T base pairs Many A-T base pairs
because easier to break 2 because easier to break 2 H-bonds that 3 H-bondsH-bonds that 3 H-bonds
Note anti-parallel chains
Replication Fork
Bidirectional movement of the DNA replication machinery
THE REPLICATION FACTORY
DNA replication is an intricate process requiring the concerted action of many different proteins.
The replication proteins are clustered together in particular locations in the cell and may therefore be regarded as a small “Replication Factory” that manufactures DNA copies.
THE REPLICATION FACTORY
The DNA to be copied is fed through the factory, much as a reel of film is fed through a movie projector.
The incoming DNA double helix is split into two single strands and each original single strand becomes half of a new DNA double helix. Because each resulting DNA double helix retains one strand of the original DNA, DNA replication is said to be semi-conservative.
DNA REPLICATION PROTEINS
DNA replication requires a variety of proteins.
Each protein performs a specific function in the production of the new DNA strands.
Helicase, made of six proteins arranged in a ring shape, unwinds the DNA double helix into two individual strands.
Single-strand binding proteins, or SSBs, are tetramers that coat the single-stranded DNA.
This prevents the DNA strands from reannealing to form double-stranded DNA.
Primase is an RNA polymerase that synthesizes the short RNA primers needed to start the strand replication process.
DNA polymerase is a hand-shaped enzyme that strings nucleotides together to form a DNA strand.
The sliding clamp is an accessory protein that helps hold the DNA polymerase onto the DNA strand during replication.
RNAse H removes the RNA primers that previously began the DNA strand synthesis.
DNA ligase links short stretches of DNA together
to create one long continuous DNA strand.
Components of the DNA Replication
Polymerase & Proteins Coordinated
One polymerase complex apparently synthesizes leading/lagging strands simultaneously
Even more complicated in eukaryotes
STRAND SEPARATION
To begin the process of DNA replication, the two double helix strands are unwound and separated from each other by the helicase enzyme.
The point where the DNA is separated into single strands, and where new DNA will be synthesized, is known as the replication fork.
Single-strand binding proteins, or SSBs, quickly coat the newly exposed single strands. SSBs maintain the separated strands during DNA replication.
Replication Fork
Bidirectional movement of the DNA replication machinery
STRAND SEPARATION
Without the SSBs, the complementary DNA strands could easily snap back together.
SSBs bind loosely to the DNA, and are displaced when the polymerase enzymes begin synthesizing the new DNA strands.
NEW STRAND SYNTHESIS
Now that they are separated, the two single DNA strands can act as templates for the production of two new, complementary DNA strands.
Remember that the double helix consists of two antiparallel DNA strands with complementary 5’ to 3’ strands running in opposite directions.
NEW STRAND SYNTHESIS
Polymerase enzymes can synthesize nucleic acid strands only in the 5’ to 3’ direction, hooking the 5’ phosphate group of an incoming nucleotide onto the 3’ hydroxyl group at the end of the growing nucleic acid chain.
Because the chain grows by extension off the 3’ hydroxyl group, strand synthesis is said to proceed in a 5’ to 3’ direction.
NEW STRAND SYNTHESIS Even when the strands are separated, however,
DNA polymerase cannot simply begin copying the DNA.
DNA polymerase can only extend a nucleic acid chain but cannot start one from scratch.
To give the DNA polymerase a place to start, an RNA polymerase called primase first copies a short stretch of the DNA strand.
This creates a complementary RNA segment, up to 60 nucleotides long that is called a primer.
NEW STRAND SYNTHESIS Now DNA polymerase can copy the DNA strand.
The DNA polymerase starts at the 3’ end of the RNA primer, and, using the original DNA strand as a guide, begins to synthesize a new complementary DNA strand.
Two polymerase enzymes are required, one for each parental DNA strand.
Due to the antiparallel nature of the DNA strands, however, the polymerase enzymes on the two strands start to move in opposite directions.
NEW STRAND SYNTHESIS One polymerase can remain on its DNA
template and copy the DNA in one continuous strand.
However, the other polymerase can only copy a short stretch of DNA before it runs into the primer of the previously sequenced fragment.
It is therefore forced to repeatedly release the DNA strand and slide further upstream to begin extension from another RNA primer.
NEW STRAND SYNTHESIS The sliding clamp helps hold this DNA
polymerase onto the DNA as the DNA moves through the replication machinery. The sliding clamp makes the polymerase processive.
The continuously synthesized strand is known as the leading strand, while the strand that is synthesized in short pieces is known as the lagging strand.
The short stretches of DNA that make up the lagging strand are known as Okazaki fragments.
THE LAGGING STRAND
Before the lagging-strand DNA exits the replication factory, its RNA primers must be removed and the Okazaki fragments must be joined together to create a continuous DNA strand.
The first step is the removal of the RNA primer.
THE LAGGING STRAND
RNAse H, which recognizes RNA-DNA hybrid helices, degrades the RNA by hydrolyzing its phosphodiester bonds. Next, the sequence gap created by RNAse H is then filled in by DNA polymerase which extends the 3’ end of the neighboring Okazaki fragment.
Finally, the Okazaki fragments are joined together by DNA ligase that hooks together the 3’ end of one fragment to the 5’ phosphate group of the neighboring fragment in an ATP- or NAD+-dependent reaction.
REPLICATION IN ACTION
The process begins when the helicase enzyme unwinds the double helix to expose two single DNA strands and create two replication forks.
DNA replication takes place simultaneously at each fork. The mechanism of replication is identical at each fork.
How is DNA Synthesized?
Original theory Begin adding nucleotides at origin Add subsequent bases following pairing rules
Expect both strands to be synthesized simultaneously This is NOT how it is accomplished
How is DNA Synthesized?
Actually how DNA is synthesized Simple addition of nucleotides along one
strand, as expected Called the leading strandleading strand DNA polymerase reads 3’ 5’ along the
leading strand from the RNA primer Synthesis proceeds 5’ 3’ with respect to
the new daughter strand
Remember how the nucleotides are added!!!!! 5’ 5’ 3’ 3’
Mistakes during Replication Base pairing rules must be maintained
Mistake = genome mutation, may have consequence on daughter cells
Only correct pairings fit in the polymerase active site
If wrong nucleotide is included Polymerase uses its proofreadingproofreading ability to
cleave the phosphodiester bond of improper nucleotide Activity 3’ 5’
And then adds correct nucleotide and proceeds down the chain again in the 5’ 3’ direction
Proofreading
DNA Repair
For the rare mutations occurring during replication that isn’t caught by DNA polymerase proofreading
For mutations occurring with daily assault
If no repair In germ (sex) cells inherited diseases In somatic (regular) cells cancer
CONSEQUENCES OF GENETIC ERRORS :SOURCES OF GENETIC VARIATION
Mutation - any novel genetic change in the gene complement or genotype relative to the parental genotypes, beyond that achieved by genetic recombination during meiosis.
Mutations are changes in DNA structure, and therefore changes in protein and phenotype.
CONSEQUENCES OF GENETIC ERRORS SOURCES OF GENETIC VARIATION
Mutations are rare! For every 100 million nucleotides added to a developing DNA strand only one mistake occurs on average.
Mutations are heritable; and may be beneficial, neutral, lethal, detrimental or harmful to the organism.
Types of Mutation
1. Induced viruses, UV radiation, some
chemicals (nitric acid changes cytosine to uracil) or mutagens (or carcinogens - benzene, cigarette smoke).
Types of Mutation
2. Spontaneous Proofreading mistakes during DNA
replication (Base substitutions) - not necessarily a serious change.
Frame shift mutation (Addition or deletion of a base) - serious change!
Types of Mutation
A 3 letter code or codon is analogous to three letter words in a sentence.
Original sequence
THE CAT SAW THE DOG
Base or letter substitutions
THE BAT SAW THE DOG
THE CAT SAW THE HOG
THE CAB SAW THE DOG
THE CAT SAW SHE DOG
THE CAT SAD THE DOG
THE CAT SAW THE DOC
Types of Mutation
Deletions
THE CAT SAW TED OG
THE ATS AWT HED OG
Additions
THE CAT SAW THE ZDO G
THE CMA TAS WTH EDO G
Types of Mutation
3. Jumping genes, transposable elements, or transposons.
Discovered by Barbara McClintok (1956) while studying color variation in Indian corn.
Won Nobel prize in 1983.
Types of Mutation
3. Jumping genes, transposable elements, or transposons.
Patches of yellow sometimes occur among the purple grains of Indian corn. She explain this by assuming that the gene was being interrupted by a foreign sequence of DNA.
These foreign bits of DNA could insert or remove themselves from a stretch of DNA causing the genes that they affected to be turned on or off. Such "jumping genes" could copy themselves and move about within the genome of the organism they occupied.
Types of Mutation
4. Chromosomal mutations (disruption in chromosomal morphology - inversions and translocations).
5. Homeotic genes master genes that regulate suites of other
genes and may affect developmental pathways especially during embryogenesis. Mutations in these master genes can cause genetic anomalies. For example, a fruit fly that possesses legs where antennae should be, or a mosquito that has its mouth parts transformed into legs.
Effect of Mutation
Uncorrected Replication Errors
Mismatch repair Enzyme complex recognizes mistake and
excises newly-synthesized strand and fills in the correct pairing
Mismatch Repair – cont’d
Eukaryotes “label” the daughter strand with nicks to recognize the new strand Separates new
from old
Chemical Modifications
Thymine Dimers
Caused by exposure to UV light 2 adjacent thymine residues
become covalently linked
Repair Mechanisms Different enzymes
recognize, excise different mistakes
DNA polymerase synthesizes proper strand
DNA ligase joins new fragment with the polymer
STEP 2 - TRANSCRIPTION
Transcription
The region of the double-stranded DNA corresponding to a specific gene is copied into an RNA molecule, called messenger RNA (mRNA).
RNA differs from DNA Ribose is the sugar rather than
deoxyribose – ribonucleotidesribonucleotides U instead of T; A, G and C the same Single stranded
Can fold into a variety of shapes that allows RNA to have structural and catalytic functions
RNA Differences
RNA Differences
Transcription
Similarities to DNA replication Open and unwind a portion of the DNA 1 strand of the DNA acts as a template Complementary base-pairing with DNA
Differences RNA strand does not stay paired with
DNA DNA re-coils and RNA is single stranded
RNA is shorter than DNA RNA is several 1000 bp or shorter whereas
DNA is 250 million bp long
RNA Polymerase
Catalyzes the formation of the phosphodiester bonds between the nucleotides (sugar to phosphate)
Uncoils the DNA, adds the nucleotide one at a time in the 5’ to 3’ fashion
Uses the energy trapped in the nucleotides themselves to form the new bonds
Template to Transcripts
The RNA transcript is identical to the NON-template strand with the exception of the T’s becoming U’s
RNA Elongation
Reads template 3’ to 5’
Adds nucleotides 5’ to 3’ (5’ phosphate to 3’ hydroxyl)
Synthesis is the same as the leading strand of DNA
Differences in DNA and RNA Polymerases
RNA polymerase adds ribonucleotides not deoxynucleotides
RNA polymerase does not have the ability to proofreadproofread what they transcribe
RNA polymerase can work without a primer
RNA will have an error 1 in every 10,000 nucleotides (DNA is 1 in 100,000,000 nucleotides)
Types of RNA
messenger RNA (mRNA)messenger RNA (mRNA) – codes for proteins
ribosomal RNA (rRNA)ribosomal RNA (rRNA) – forms the core of the ribosomes, machinery for making proteins
transfer RNA (tRNA)transfer RNA (tRNA) – matches code for amino acid on mRNA and positions the right amino acid in place during protein synthesis
How does the process of transcription begin? The DNA serves as the template for
producing an RNA transcript or copy of information stored on the DNA molecule.
The DNA molecule must open up and allow an enzyme called RNA polymerase read and connect together the sequence of nucleotides in the proper order.
STEP 3 – TRANSLATION
RNA to Protein
Translation is the process of turning mRNA into protein
Translate from one “language” (mRNA nucleotides) to a second “language” (amino acids)
Genetic codeGenetic code – nucleotide sequence that is translated to amino acids of the protein
DNA Code
Nucleotides read 3 at a time meaning that there are 64 combinations for a codoncodon (set of 3 nucleotides)
Only 20 amino acids More than 1 codon per AA – degenerate
code with the exception of Met and Trp (least abundant AAs in proteins)
Reading Frames
Translation can occur in 1 of 3 possible reading frames, dependent on where decoding starts in the mRNA
Transfer RNA Molecules
Translation requires an adaptoradaptor molecule that recognizes the codon on mRNA and at a distant site carries the appropriate amino acid
Intra-strand base pairing allows for this characteristic shape
AnticodonAnticodon is opposite from where the amino acid is attached
Wobble Base Pairing
Due to degenerate code for amino acids some tRNA can recognize several codons because the 3rd spot can wobble or be mismatched
Allows for there only being 31 tRNA for the 61 codons
Attachment of AA to tRNA
Aminoacyl-tRNA synthaseAminoacyl-tRNA synthase is the enzyme responsible for linking the amino acid to the tRNA
A specific enzyme for each amino acid and not for the tRNA
2 ‘Adaptors’ Translate Genetic Code to Protein
1
2
Ribosomes Complex machinery that controls protein synthesis
2 subunits 1 large – catalyzes the
peptide bond formation 1 small – binds mRNA and
tRNA
Contains protein and RNA rRNA central to the
catalytic activity Folded structure is highly
conserved
Protein has less homology and may not be as important
Ribosome Structures
May be free in cytoplasm or attached to the ER Subunits made in the nucleus in the nucleolus
and transported to the cytoplasm
Ribosomal Subunits
1 large subunit – catalyzes the formation of the peptide bond
1 small subunit – matches the tRNA to the mRNA Moves along the mRNA adding amino acids to
growing protein chain
Ribosomal Movement
4 binding sites mRNA binding site Peptidyl-tRNA binding site (P-site)
Holds tRNA attached to growing end of the peptide
Aminoacyl-tRNA binding site (A-site) Holds the incoming AA
Exit site (E-site)
E-site
Summary