Bio 107 Ch. 25: DNA Structure and Gene Expression

What must genetic material be able to do?

• store genetic information

• be replicated properly

• undergo mutations

• control cell activities

Frederick Griffith (1932)

researched the genetic role of DNA with streptococcus pneumoniae (used 2 strains)

• S Strain: smooth with capsule and virulent (causes dz)

• R Strain: rough and lacks capsule, nonvirulent

Frederick Griffith Experiment Results

• Live S Strain = mouse dies

• Live R Strain = mouse lives

• Heat-killed S Strain = mouse lives

• Live R Strain + Heat-killed S Strain = mouse dies

• Conclusion: some genetic material was transferred from dead S Strain to live R Strain making it virulent (transformation)

Transformation

a change in genotype and phenotype due to assimilation of foreign DNA

Avery, Mac-Leod, and McCarty (1944)

researched what the “transforming substance” (DNA) was by extracting material from the heat-killed S Strain and treating it with enzymes 

Avery, Mac-Leod, and McCarty Experiment Results

• DNase: destroyed DNA and transformation did NOT occur

• RNase: destroyed RNA and transformation still occurred

• Protease: destroyed proteins and transformation still occurred

• Conclusion: DNA was the “transforming substance” and carries genetic material 

Hersey and Chase (1952)

confirmed whether DNA or protein was the genetic material of viruses by injecting a virus’ genetic material into host cell (E. Coli)

Hersey and Chase Experiment Results

• Viral DNA (32P) = entered bacteria

• Viral Protein (35S) = stayed in medium and did NOT enter bacteria

• Conclusion: only DNA enters and carries genetic information

Who determined the structure of DNA?

James Watson and Francis Crick in 1953

DNA

a polynucleotide (a chain of nucleotides) with a backbone of alternative phosphate and sugar groups

• double helix shape

Nucleotides

a complex of 3 subunits

• Phosphoric Acid (phosphate)

• A pentose sugar (deoxyribose)

• A nitrogen-containing base

4 Possible DNA Bases

2 Purins: Adenine (A) and Guanine (G)

2 Pyrimidines: Thymine (T) and Cytosine (C)

• A + T connected by 2 hydrogen bonds

• C + G connected by 3 hydrogen bonds

Double Helix of DNA

2 strands held together by hydrogen bonds between bases

• the strands are antiparallel-oriented in opposite directions

• 5’ carbon is the uppermost on one strand and 3’ carbon on the other uppermost 

DNA Replication

copying 1 DNA double helix into 2 identical double helixes

DNA Polymerase

the key enzyme in DNA replication that catalyzes the elongation of new DNA bonds using helicase

• joins and positions the new matching DNA nucleotides

Helicase

an enzyme that unwinds and unzips DNA by breaking the hydrogen bonds

Semiconservative Replication

each original DNA strand is uses as a template to produce a new complimentary strand

• each daughter DNA consists of one new chain of nucleotides and one identical from parent DNA

What direction does DNA strands occur in?

opposite directions because the strands are antiparallel and DNA Polymerase can only add new nucleotides

• Leading Strand: follows DNA helicase

• Lagging Strand: synthesized in Okazaki fragments

Lagging Strand and DNA Ligase

DNA Ligase connects the okazaki fragments and seals the break in the sugar-phosphate backbone

Replication Fork

the unwinding of DNA strand create a single-stranded DNA that serves as a template for replication and moves unidirectional in a “zipper-like” fashion

Steps of DNA Replication

1. Before replication, parent molecule strands are hydrogen-bonded

2. DNA Helicase unzips double helix

3. DNA Polymerase adds complementary nucleotides to each strand

4. DNA Ligase seals gaps in the sugar-phosphate backbone

5. 2 identical DNA molecules are formed

Gene

a segment of DNA or genomic sequence (DNA or RNA) that directly codes functional products (RNA or proteins)

Gene Expression

the process of using a gene sequence to synthesize a protein that depends on 3 different types of RNA

• Messenger RNA (mRNA)

• Transfer RNA (tRNA)

• Ribosomal RNA (rRNA)

What are the 2 processes gene expression depends on?

• Transcription: takes place in the nucleus and part of the DNA serves as a template for mRNA formation

• Translation: takes place in cytoplasm in which a sequence off mRNA bases determine the sequence of amino acids in a polypeptide

Transciption

a gene serving as a template to produce an RNA molecule, transferring genetic information from DNA to RNA

Messenger RNA (mRNA)

a copy of a sequences of bases containing codons that serves to carry genetic information from DNA to ribosome for protein synthesis

• formation begins when RNA Polymerase binds to a promotor in DNA

Promotor

a specific DNA sequence that acts like a “start” signal for a gene

RNA Polymerase

joins new complementary RNA nucleotides using U, A, C, or G

Pre-mRNA

the first RNA molecule made and is not ready to leave nucleus

• contains complementary bases, introns, and exons

Introns

noncoding regions that need to be removed

Exons

coding regions used to produce proteins by joining to form a mature mRNA

Matura mRNA

ready when a guanine cap is added to the 5’ end and a poly-A tail added to 3’ end

RNA Splicng

introns removed and exons joined

The Genetic Code

translate the 3-letter codons on mRNA into amino acids and is UNIVERSAL

Codons

3-base “words” that each specify to one amino acid

Triplet Codon

each codon = 3 RNA bases (ex. AUG, CCG, UAA)

How many different mRNA codons are there?

64

• 61 code for certain amino acids, may lead to a redundant code

• 3 are stop codons

Redundant Code

multiple codons can code for the same amino acid and may provide some protection against mutations

Stop Codons

UAA, UAG, and UGA = signal translation to stop

Start Codon

AUG (methionine) = signal where translation begins

Translation

the 2nd process for protein synthesis that decodes a mRNA sequence into a chain of amino acids using all 3 RNA molecules

Transfer RNA (tRNA)

transports the correct amino acids to the ribosome by reading the mRNA codon

Structure of tRNA

cloverleaf shape in which one end binds to a specific amino acid and the other end containg a 3-base anticodon

Anticodon

a triplet code complementary to a specific mRNA codon

What determines the order in which tRNA brings amino acids?

the order of mRNA codons

Ribosomes

made up of rRNA and proteins and either exist freely OR attached to endoplasmic reticulum

What are the 2 subunits that mist bind before translation?

• Small Subunit: binds mRNA

• Large Subunit: binds tRNA and forms peptide bonds

Binding Site

where complementary base pairing between anticodons and codons occurs and where polypeptides from

Polypeptide

specific order of amino acids

Polyribosome

a complex formed when other ribosomes attach to mRNA and polypeptides are copied

3 Binding Sites of tRNA

• A Site (Amino Acid): where new tRNA enters

• P Site (Peptide): holds growing peptide chain

• E Site (Exit): where tRNA leaves ribosome

Translation Steps

• Initiation

• Elongation

• Termination

Initiation

brings all transition components, or initiation factors, together with a signal from start codon (AUG)

Initiation Factors

• Small Ribosomal Subunit: attaches to mRNA

• Initiator tRNA (UAC): attaches to start codon on mRNA

• Large Ribosomal Subunit: joins to small subunits formaing a complete ribosome

Elongation

the polypeptide chain increase in length one amino acid at a time

Elongation Factors

required for binding between anticodons and codons

Elongation Steps

1. new tRNA amino acids enter A Site

2. peptide bonds form with growing chain in P Site

3. ribosome shifts (translocation) one codon down and empty tRNA exits from E Site

Termination

when a stop codon (UAA, UAG, UGA) enters A Site and a release factor binds instead because there’s no matching tRNA

• ribosomes detach back into 2 subunits

• polypeptide becomes a functional protein

Release Factor

binds to A site instead of tRNA causing the release of polypeptide chain

Gene Expression

how a cell turns genes on/off in order to…

• save energy (not make unnecessary proteins)

• respond to environment

• differentiate (specialized cell functions)

• maintain homeostasis

Housekeeping Genes

genes that are always active (expressed)  to perform basic, common functions

Gene Expression in Prokaryotes

prokaryotes (ex. E. Coli) control genes by turning transcription on/off

E. Coli

uses various sugars as an energy source and can adjust its gene expression based on available sugar

Operon

a cluster of genes working together under a promotor, acting as an on/off switch

Parts of an Operon

• Promotor

• Operator

• Structural Genes

• Regulatory Gene

Promotor

DNA region where RNA Polymerase binds to start transcription

Operator

the “switch” that controls whether transcription happens and where the repressor binds

Types of Operons

• Inducible Operon: normally off but turned ON when a molecule, like lactose, is present (ex. Iac Operon)

• Repressible Operon: normally on but turned OFF when a specific molecules activates it

Iac Operon (E. Coli)

controls genes that break down lactose (milk sugar); an inducible operon

Iac Repressor

encoded by a regulatory gene located outside the operon

What if lactose is ABSENT?

the operon is OFF and the Iac Repressor binds to the operator blocking RNA Polymerase from making enzymes since there is no lactose to digest

What if lactose is PRESENT?

the operon is ON and lactose binds to the Iac Repressor changing its shape making it unable to bind to operator so RNA Polymerase can make lactose-digesting enzymes 

Gene Expression in Eukaryotes

much more complex as each gene has its own promoter and employs various mechanisms to control

• determines IF a gene is expressed, how FAST its expressed, and how LONG it stays active

Levels of Gene Control in Eukaryotes

• Pretranscriptional Contro

• Transcriptional Control

• Transcriptional Control

• Translational

• Post-Translational

Pre-transcriptional Control

determines if DNA is accessible for transcription and uses DNA Methylation and chromatin packing to keep genes OFF

• tightly coiled chromatin = transcription off (heterochromatin)

• loose chromatin = transcription ON (euchromatin)

Heterochromatin

tightly packed, dark-staining regions where inactive genes are found

Euchromatin

loosely packed, light regions where active genes are found

Chromatin Remodeling Complex

pushes aside nucleosomes so RNA Polymerase can access DNA

Transcriptional Control

controls how often transcription begins depending on transcription factors

Transcription Factors

bind to either promotor or enhancers to either to activate (turn transcription ON) or repress (turn transcription OFF)

Promotor and Enhancers

DNA sequences proteins attach to

Post-Transcriptional Control

regulates how mRNA is made before it can leave the nucleus

• where primary mRNA is processed into mature mRNA by alternative splicing

Alternative Splicing

splicing different exons together and removing introns to make proteins

Translational Control

controls when and how often translation occurs affected by different factors…

• longer time mRNA lasts in cytoplasm = more proteins made

• poly-A tail length = mRNA stability

Post-Transitional Control

regulates protein activation and its lifespan, but some may need activation using chemical modifcations 

Chemical Modifications

can turn proteins on/off (ex. phosphorylation)

Gene Mutations

a permanent change in DNA base sequences which may change gene expression or make a protein nonfunctional/inactive

Type of Mutations

• Germ-Line Mutations

• Somatic Mutations

Germ-Line Mutation

occur in sex cells and be passed down including cancer or genetic disorders

Somatic Mutations

occur in body cells and NOT passed down but can also lead to cancer

Causes of Mutations

• Spontaneous Mutation

• Induc Mutation

• Error in DNA Replication

• Mutagens

• Transposons

Spontaneous Mutation

due to abnormalities in normal biological processes

Induced Mutations

due to environmental influences

Errors in DNA Replication

occurs rarely because there is proofreading to minimizes errors in new strands

Mutagens

include environmental influences like radiation, x-ray, UV, cigarette smoke, and pesticides

DNA Repair Enzymes

constantly monitors and repairs irregularities keeping mutagen rate LOW

Transposons

jumping genes that move within and between chromosomes that may alter neighbouring gene expression

Point Mutations

involve a change in a single DNA nucleotide

Possible Outcomes of Mutations on Proteins

• change in specific amino acid

• NO effect

• abnormal protein (ex. sickle cell)

• incomplete proteins

Frameshift Mutation

1 or more nucleotides are either inserted or deleted from DNA resulting in a new codon sequence and nonfunctional protein