molecular biology: replication,...
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MOLECULAR BIOLOGY: REPLICATION, TRANSCIPTION,
AND TRANSLATIONHonors Biology
2012
IMPORTANT EXPERIMENTS
• Frederick Griffith
• Described a “transforming factor” that could be transferred into a bacterial cell
• Process is called transformation
IMPORTANT EXPERIMENTS
• Hershey-Chase
• Used bacteriophages to show that DNA is the genetic material
• Phages were labeled with radioactive sulfur (labels proteins) and radioactive phosphorus (labels DNA)
• Sulfur-labeled protein stayed outside the cell
• Phosphorus-labeled DNA was inside the cell
Head
Tail fiber
DNA
Tail
Batch 1 Radioactive protein
Bacterium
Radioactive protein
DNA
Phage
Pellet
Radioactive DNA Batch 2
Radioactive DNA
Empty protein shell
Phage DNA
Centrifuge
Radioactivity in liquid
Measure the radioactivity in the pellet and the liquid.
4 Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube.
3 Agitate in a blender to separate phages outside the bacteria from the cells and their contents.
2 Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells.
1
Pellet
Centrifuge Radioactivity in pellet
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2
3
NUCLEOTIDES • Monomer of DNA and RNA is
called a nucleotide
• Contains three parts: nitrogenous base, 5-carbon sugar, and phosphate group
• DNA and RNA are called polynucleotides
• Composed of a sugar-phosphate backbone
• Nitrogenous bases point inward
• Four DNA bases: thymine, cytosine, adenine, and guanine
Sugar-phosphate backbone
DNA nucleotide
Phosphate group
Nitrogenous base Sugar
DNA polynucleotide
DNA nucleotide
Sugar (deoxyribose)
Thymine (T)
Nitrogenous base (A, G, C, or T)
Phosphate group
Pyrimidines
Guanine (G) Adenine (A) Cytosine (C) Thymine (T)
Purines
WATSON AND CRICK• Credited with
discovering the structure of DNA
• Really took ideas from others and were the first to publish
• Took X-ray crystallography data from Rosalind Franklin without her knowledge
DNA STRUCTURE• Composed of two
polynucleotide chains joined together by hydrogen bonds between bases
• A pairs with T, forming two hydrogen bonds
• G pairs with C, forming three hydrogen bonds
• Twisted as a helical shape
Hydrogen bond
Base pair
Partial chemical structure Computer model Ribbon model
Twist
4
5
6
DNA REPLICATION
• Semiconservative model
• Two DNA strands separate
• Each strand is used as a pattern to produce a complimentary strand
• Each new DNA helix has one old strand and one new strand
Parental molecule of DNA
Nucleotides
Both parental strands serve as templates
Two identical daughter
molecules of DNA
DNA REPLICATION• Replication occurs in the 5’ → 3’
direction
• Leading strand = continuous replication
• Lagging strand = segmented replication
• Enzymes important for replication
• DNA helicase binds to unwind the DNA
• DNA polymerase adds new base pairs
• DNA ligase joins fragments together
Origin of replication Parental strand Daughter strand
Bubble
Two daughter DNA molecules
3! end 5! end
3! end 5! end
3!
5!
2!
4! 1! 3!
5!
2!
4! 1!
P
P
P
P
P
P
P
P
Parental DNA 3! 5!
DNA polymerase molecule
DNA ligase
3! 5!
Overall direction of replication
Daughter strand synthesized continuously
3! 5!
3! 5!
Daughter strand synthesized in pieces
FROM DNA TO PROTEIN
• Gene sequence of DNA directs for the synthesis of a protein
• DNA is transcribed into RNA
• RNA is translated into protein
Cytoplasm
Nucleus
DNA
Transcription
RNA
Translation
Protein
Polypeptide
Translation
Transcription
DNA strand
Codon
Amino acid
RNA
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8
9
GENETIC CODE• Codons - three nucleotide
sequences that correspond to a particular amino acid
• 61 codons code for an amino acid (more than one codon can code for an amino acid)
• AUG - start codon (codes for the amino acid methionine)
• Three stop codons (UAA, UAG, and UGA)
Firs
t bas
e
Thir
d ba
se
Second base
TRANSCRIPTION• DNA strands separate
• One strand is used as a patter to produce RNA
• Important change: In RNA thymine (T) is not used; uracil (U) is used instead.
• RNA polymerase is the enzyme that complete transcription
• Three stages:
• Initiation: RNA polymerase binds to a promoter to unwind the helix
• Elongation: RNA nucleotides are added to the chain
• Termination: RNA polymerase reaches a terminator sequence and detaches
RNA polymerase
Newly made RNA
Direction of transcription Template
strand of DNA
RNA nucleotides
Terminator DNA
DNA of gene
RNA polymerase
Initiation
Promoter DNA
1
Elongation 2
Area shown in Figure 10.9A
Termination 3 Growing
RNA
RNA polymerase
Completed RNA
RNA PROCESSING• Messenger RNA (mRNA) - contains
codons for protein sequences
• Introns - interrupting segments
• Exons - coding segments
• Processing
• Cap added to the 5’ end (guanine nucleotides)
• Tail added to the 3’ end (Poly-A tail - 50-250 adenines)
• RNA Splicing - removal of introns and joining of exons
RNA transcript with cap and tail
Exons spliced together
Introns removed
Transcription Addition of cap and tail
Tail
DNA
mRNA
Cap
Exon Exon Exon Intron Intron
Coding sequence Nucleus
Cytoplasm
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11
12
TRANSLATION
• Transfer RNA (tRNA) - match amino acids with the mRNA codon
• Each tRNA carries a specific amino acid
• Anticodon allows tRNA to bind to the complimentary codon on mRNA (A pairs with U; G pairs with C)
Anticodon
Amino acid attachment site
RNA polynucleotide chain
Hydrogen bond
TRANSLATION• Occurs at the ribosome (rRNA) in the
cytoplasm
• Three stages:
• Initiation: mRNA binds to ribosome and first tRNA at the start codon (P site)
• Elongation: next tRNA binds to mRNA at the A site and the two amino acids are joined as the ribosomes moves the molecules over to the next site
• Termination: stop codon is reached, all of the components are released
Polypeptide
A site
1 Codon recognition Codons
Amino acid
Anticodon P site
mRNA
2 Peptide bond formation
3 Translocation
New peptide bond
Stop codon
mRNA movement
Small ribosomal subunit
Start codon
P site
mRNA
A site
Large ribosomal subunit
Initiator tRNA
Met Met
2 1
OVERVIEW OF TRANSCRIPTION AND TRANSLATION
mRNA is transcribed from a DNA template.
RNA polymerase
Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP.
Amino acid
DNA Transcription
mRNA
tRNA ATP
Translation
Enzyme
The mRNA, the first tRNA, and the ribosomal sub-units come together.
Initiator tRNA
Large ribosomal subunit
Anticodon
Initiation of polypeptide synthesis
Small ribosomal subunit
mRNA
Start Codon
1
2
3
New peptide bond forming Growing
polypeptide
4
A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time.
Elongation
Codons
mRNA
Polypeptide
5 The ribosome recognizes a stop codon. The polypeptide is terminated and released.
Termination
Stop codon
13
14
15
MUTATIONS• Mutation - change in nucleotide
sequence
• Base substitutions: replacement of one nucleotide with another
• Could have no effect or would effect only one amino acid
• Deletions or insertions
• Can alter the reading frame and thus have significant impacts downstream
• Can be spontaneous (due to errors in replication or recombination) or induced by mutagens (radiation or chemicals)
Normal hemoglobin DNA Mutant hemoglobin DNA
Sickle-cell hemoglobin Normal hemoglobin
mRNA mRNA
Val Glu
Substitutions
Normal gene
Protein
Base substitution
Base deletion Missing
mRNA
Met Lys Phe Ser Ala
Met Lys Phe Gly Ala
Met Lys Leu Ala His
Insertions or Deletions
MICROBIAL GENETICS: VIRUSES• Two types of
reproductive cycles:
• Lytic: viral particles are produced using host cell components and the host cell dies when the viruses are released
• Lysogenic: viral DNA is inserted into the host by recombination
Bacterial chromosome
Phage injects DNA
Phage
Phage DNA
Attaches to cell
2
1
3
Phage DNA circularizes
Lytic cycle
4
New phage DNA and proteins are synthesized
Phages assemble
Cell lyses, releasing phages
6 5
7
Phage DNA inserts into the bacterial chromosome by recombination
Lysogenic bacterium reproduces normally, replicating the prophage at each cell division
Prophage
Lysogenic cycle
Many cell divisions
OR
ENVELOPED VIRUSES
Plasma membrane of host cell
VIRUS
Entry
Uncoating
Viral RNA (genome)
Viral RNA (genome)
2
1
3
Membranous envelope
Protein coat Glycoprotein spike
RNA synthesis by viral enzyme
Template
RNA synthesis (other strand)
Protein synthesis
mRNA
4 5
6
New viral genome
New viral proteins
Assembly
7
Exit
16
17
18
RETROVIRUSES
• HIV (human immunodeficiency virus)
• Contains two copies of its RNA genome and an enzyme, reverse transcriptase, which can produce DNA from an RNA template
Reverse transcriptase
RNA (two identical strands)
Protein coat
Glycoprotein Envelope
Double- stranded DNA
Viral RNA and proteins
DNA strand
Viral RNA
NUCLEUS
CYTOPLASM
Chromosomal DNA
Provirus DNA
RNA
2
1
5
3
4
6
VIROIDS AND PRIONS
• Viroid - circular RNA that infects plants
• Prions - infectious proteins that cause nervous system disorders in animals
• Misfolded forms of normal proteins that can convert other proteins
BACTERIA• Three ways they transfer
DNA
• Transformation - uptake of DNA from the environment
• Transduction - gene transfer through bacteriophages
• Conjugation - transfer of DNA from a donor to a recipient through a cytoplasmic bridge
Phage
Fragment of DNA from another bacterial cell (former phage host)
DNA enters cell
Bacterial chromosome (DNA)
Fragment of DNA from another bacterial cell
Mating bridge
Sex pili
Donor cell (“male”)
Recipient cell (“female”)
Transformation
Transduction
Conjugation
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PLASMIDS
• Small circular DNA molecules that are separate from the bacterial chromosome
Male (donor) cell
Bacterial chromosome
F factor starts replication and transfer
F factor (plasmid)
Plasmid completes transfer and circularizes
Cell now male
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