Download Bio 103 Lecture - Molecular Biology of t

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Molecular Biology of the Gene
Bio 103 Lecture
Dr. Largen
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4Finding the genetic material
4Structure of the genetic material
4DNA replication
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4Flow of genetic information from DNA to RNA to protein
Search for genetic material lead to DNA
Search for genetic material lead to DNA
4 DNA was identified as a substance in cells 100 years ago
– but scientists had no knowledge of its role in heredity at that time
4 During the 1920’s-1950’s, experimental evidence mounted that DNA was the genetic
material rather than protein
– protein and DNA are both complex macromolecules found in chromosomes
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Search for genetic material lead to DNA
4 Experiments
in early 1900s had shown that genes were located on chromosomes
– two constituents of chromosomes were
• proteins
• DNA
4 therefore, the two candidates for genetic material were
– protein
– DNA
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Search for genetic material lead to DNA
4 Until 1940s
– the great heterogeneity and specificity of function of proteins indicated that proteins were the
genetic material.
4 However, this was not consistent with experiments with microorganisms, like bacteria and
viruses.
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Search for genetic material lead to DNA
4 Discovery of the genetic role of DNA began with research by Frederick Griffith in 1928.
– was trying to develop vaccine for pneumonia in humans
– studied Streptococcus pneumoniae
• bacterium that causes pneumonia in mammals.
– One strain, the R strain, was harmless.
– other strain, the S strain, was pathogenic.
– mixed heat-killed S strain with live R strain bacteria and injected this into a mouse.
• mouse died and he recovered the pathogenic strain from the mouse’s blood.
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Search for genetic material lead to DNA
4 Griffith concluded that
– the live cells had been
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• transformed
– transformation is a change in genotype and phenotype due to the assimilation of a
foreign substance (now known to be DNA) by a cell.
– in the presence of the dead S cells
• some of the living R cells had been transformed into virulent S strain organisms
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Search for genetic material lead to DNA
4 Griffith concluded that
– some chemical component of the dead pathogenic cells, some “transforming factor” caused
• the heritable change
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Search for genetic material lead to DNA
4 For
next 14 years scientists tried to identify the “transforming factor”.
4 in 1944
– Oswald Avery, Maclyn McCarty and Colin MacLeod announced that “transforming factor”
was DNA
4 Still, many biologists were skeptical.
– In part, this reflected a belief that the genes of bacteria could not be similar in composition
and function to those of more complex organisms.
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Search for genetic material lead to DNA
4 Further
evidence that DNA was the genetic material was
– derived from studies that tracked the infection of bacteria by viruses.
4 Viruses
– consist of a DNA (sometimes RNA) enclosed by a protective coat of protein.
– To replicate, a virus infects a host cell and takes over the cell’s metabolic machinery.
4 Viruses that specifically attack bacteria are called bacteriophages or just phages.
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Search for genetic material lead to DNA
4 In 1952
– Alfred Hershey and Martha Chase showed that DNA was the genetic material of
• T2 phage
– consists almost entirely of DNA & protein
– attacks Escherichia coli (E. coli)
» common intestinal bacteria of mammals.
– can quickly turn E. coli cell into a T2-producing factory that
» releases phages when cell ruptures.
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Search for genetic material lead to DNA
4 Hershey
and Chase experiment
– designed to determine the source of genetic material in the phage
– consisted of
• radioactively labeled protein andr DNA
• track which entered the E. coli cell during infection.
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Search for genetic material lead to DNA
4 Hershey
and Chase experiment
– grew one batch of T2 phage in the presence of radioactive sulfur
• marking the proteins but not DNA.
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– Sulfur part of some amino acids
– grew another batch in the presence of radioactive phosphorus
• marking the DNA but not proteins
– phosphorous is part of nucleotides
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Search for genetic material lead to DNA
4 Hershey
and Chase experiment
– allowed each batch to infect separate E. coli cultures.
• Shortly after the onset of infection
– they spun the cultured infected cells in a blender, shaking loose any parts of the
phage that remained outside the bacteria.
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Search for genetic material lead to DNA
4 Hershey
and Chase experiment
– mixtures were spun in a centrifuge
• which separated
– heavier bacterial cells in the pellet from
– lighter free phages and parts of phage in the liquid supernatant.
– tested the pellet and supernatant of the separate treatments for the presence of radioactivity.
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Search for genetic material lead to DNA
4 Hershey
and Chase experiment
– found that when the bacteria were
• infected with T2 phages with radio-labeled proteins
– most of the radioactivity was in the supernatant, not in the pellet.
• infected with T2 phages with radio-labeled DNA
– most of the radioactivity was in the pellet with the bacteria
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Search for genetic material lead to DNA
4 Hershey and Chase
– concluded
• phage DNA was injected into host cell
• most of phage protein remained outside
– showed that
• the injected DNA molecules cause the cells to produce additional phage DNA and protein
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DNA and RNA are polymers of nucleotides
DNA and RNA are polymers of nucleotides
4nucleic acids
– two types
• DNA
– deoxyribonucleic acid
• RNA
– ribonucleic acid
– are the information storage devices of cells
– long polymers (polynucleotide) of repeating subunits (or monomers) called
nucleotides
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DNA and RNA are polymers of nucleotides
4nucleotides
• consist of three components
– a five-carbon sugar
» ribose in RNA
» deoxyribose in DNA
– a phosphate group
– a nitrogenous base
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DNA and RNA are polymers of nucleotides
4Three components of nucleotides
– nitrogenous base
• two types of organic bases occur in nucleotides
– purines
– pyrimidines
DNA and RNA are polymers of nucleotides
4Three components of nucleotides
– nitrogenous base
• purines
– large, double-ringed molecules
» adenine (A) - found in RNA and DNA
» guanine (G) - found in RNA and DNA
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DNA and RNA are polymers of nucleotides
4Three components of nucleotides
– nitrogenous base
• pyrimidines
– smaller, single-ringed molecules
» cytosine (C) – found in RNA and DNA
» thymine (T) – found in DNA only
» uracil (U) – found in RNA only
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DNA and RNA are polymers of nucleotides
4 Nucleotides are joined together to form polynucleotides
– the sugar of one nucleotide covalently bonds to the phosphate of another nucleotide
• results in a sugar-phosphate backbone
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– nitrogenous bases protrude off the sugar-phosphate backbone
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DNA is a double-stranded helix
DNA is a double-stranded helix
4 DNA is a double helix
– two polynucleotides wrap around each other
– nitrogenous bases protrude from two sugar-phosphate backbones into center of helix
where they pair
• adenine (A) with thymine (T)
• cytosine (C) with guanine (G)
– the base pairs are “held” together with hydrogen bonds
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DNA is a double-stranded helix
4 RNA
– usually consists of a single polynucleotide strand
– serves as an intermediary for DNA
• DNA’s information is transcribed into RNA
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DNA replication depends on specific base pairing
DNA replication depends on specific base pairing
4 Even
before identification of DNA as genetic material it was proposed that gene replication was
based on concept of
– complementary surfaces
• “negative image” is created along with original “positive image”
– as clay or plaster forms negative shape when it is packed around an object
– gene’s negative image could then serve as a “template” (mold)
• for making copies of the original positive image
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DNA replication depends on specific base pairing
4 DNA is copied by specific pairing of complementary bases
– each half of the double helix functions as a template upon which a new, missing half
is built
• by applying the base pairing rules to each half
– A -T and C - G
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DNA replication depends on specific base pairing
4 DNA
replication process
– two “parental” strands of DNA separate
– each separated strand becomes a template for the assembly of a complementary strand
from a supply of free nucleotides which
• line up along the template strand according to the base-pairing rules.
• are linked to form new strands.
– produces two “daughter” DNA strands, each of which is identical to the original “parent” DNA
molecule
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DNA replication depends on specific base pairing
4 Challenges for DNA replication process
– general mechanism is conceptually simple
– actual process involves complex “biochemical gymnastics”
• helical DNA must untwist as it replicates
• both strands are copied ~ simultaneously
• process proceeds rapidly
– nucleotides added at rate of
» ~50 per second in mammals
» ~500 per second in bacteria
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DNA replication: a closer look
DNA replication: a closer look
4 replication of a DNA molecule begins at special sites on the double helix, origins of
replication.
– in bacteria, this is a single specific sequence of nucleotides that is recognized by
• replication enzymes.
– Which separate the strands, forming a replication “bubble”.
– replication proceeds in both directions until the entire molecule is copied.
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DNA replication: a closer look
4 replication
of a DNA molecule
– parental strands open up as daughter strands elongate on both sides of the bubble
4 in eukaryotes, there may be hundreds or thousands of origin sites per chromosome.
• at the origin sites, the DNA strands separate forming a replication “bubble” with
replication forks at each end.
• the replication bubbles elongate as the DNA is replicated and eventually fuse.
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DNA replication: a closer look
4 DNA’s
sugar-phosphate backbone runs in opposite directions (antiparallel)
– each strand has a
• 3’ end (“three-prime”) and 5’ end (“five-prime”)
– “primed” numbers refer to position of carbon atoms of nucleotide sugars
» 3’ end: 3’ carbon is attached to an - OH group
» 5’ end: 5’ carbon has phosphate group attached
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DNA replication: a closer look
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4 New
nucleotides are linked to a growing daughter DNA strand by enzymes called
– DNA polymerases
• add nucleotides only to the 3’ end of the strand
– never to the 5’ end
• daughter strands can grow in the 5’ to 3’ direction
4 therefore, opposite orientation of DNA strands is important in DNA replication
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DNA replication: a closer look
4 DNA polymerases
– As nucleotides align with complementary bases along the template strand
• they are added to the growing end of the new strand by the polymerase
– rate of elongation
» ~ 500 nucleotides per second in bacteria
» ~ 50 per second in human cells
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DNA replication: a closer look
4 DNA polymerases
– can only add nucleotides to 3’ end of a growing DNA strand
• new strand can only elongate in 5’ to3’ direction.
– creates problem at replication fork
• one parental strand is oriented 3’->5’ into the fork
– called the leading strand
• other oriented 5’->3’ into the fork
– called the lagging strand
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DNA replication: a closer look
4 DNA polymerases
– leading strand (oriented 3’ to 5” into fork)
• can be used by polymerases as a template for a continuous complimentary strand.
– lagging strand (oriented 5’->3’ into the fork)
• is copied away from the fork in short segments called Okazaki fragments
– each about 100-200 nucleotides long
– are then joined by DNA ligase into a single DNA strand
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DNA genotype is expressed as proteins
DNA genotype expressed as proteins
4 information content of DNA
– is in specific sequences of nucleotides
4 DNA inherited by an organism leads to specific traits by
– dictating the synthesis of proteins
• proteins are the links between genotype and phenotype.
– example, Mendel’s dwarf pea plants lack a functioning copy of the gene that specifies the
synthesis of a key protein, gibberellins
» which stimulate the normal elongation of stems.
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DNA genotype expressed as proteins
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4 transcription and
translation are the two main processes linking gene to protein
4 genes provide the instructions for making specific proteins
4 bridge between DNA and protein synthesis is RNA.
– which is chemically similar to DNA
• substitutes ribose as its sugar and nitrogenous base uracil for thymine.
• almost always consists of a single strand.
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DNA genotype expressed as proteins
4 Flow
of genetic information in a eukaryotic cell:
4 DNA
– transcription
• RNA
– translation
» protein
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Genetic information is written as codons
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Genetic information is written as codons and translated into amino acid sequences
4 Nucleotides in nucleic acids represent “letters in an alphabet”
– there are four “letters”
– the bases A, T, C, G
– that can form 20 “words”
• the 20 amino acids
• each “word” codes for one amino acid in a polypeptide (or protein)
– the smallest “words” of uniform length that can specify the 20 amino acids, consist of 3
“letters”, or bases
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Genetic information is written as codons and translated into amino acid sequences
4 Arriving
at a “word” length of 3
– a two-nucleotide codon would not yield enough combinations to code for the 20 different
amino acids that commonly occur in proteins
• with the 4 DNA nucleotides, only 42 (=16) different pairs of nucleotides were possible
• but if these 4 DNA nucleotides were arranged in combinations of 3, then 4 3 (=64)
different pairs of nucleotides were possible
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Genetic information is written as codons and translated into amino acid sequences
4 The flow of information from gene to protein is based on a triplet code
– the genetic instructions for the amino acid sequence of a polypeptide (protein) are
written in DNA and RNA as a series of three-base words, called codons
• codons in DNA are transcribed into complementary three-base codons in RNA
– the RNA codons are then translated into amino acids that form a polypeptide
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Genetic code is Rosetta stone of life
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Genetic code is Rosetta stone of life
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4 task
of matching each codon to its amino acid counterpart began in the early 1960s
– Marshall Nirenberg determined the first match
• UUU coded for amino acid phenylalanine.
– created artificial mRNA molecule entirely of uracil & added it to a test tube mixture of
amino acids, ribosomes, and other components for protein synthesis.
» this “poly(U)” translated into a polypeptide containing a single amino acid,
phenyalanine, in a long chain.
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Genetic code is Rosetta stone of life
4 task
of matching each codon to its amino acid counterpart began in the early 1960s
– by variations on Marshall’s method
• the amino acids specified by all the codons were determined
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Genetic code is Rosetta stone of life
4 By the mid-1960s the entire code was deciphered
– 61of 64 triplets code for amino acids
• codon AUG not only codes for the
amino acid methionine but also indicates the start of translation
• three codons do not indicate amino
acids but signal the termination of translation
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Genetic code is Rosetta stone of life
4 genetic
code is redundant but not ambiguous.
– there are typically several different codons that would indicate a specific amino acid.
– however, any one codon indicates only one amino acid.
• If you have a specific codon, you can be sure of the corresponding amino acid, but if you
know only the amino acid, there may be several possible codons
– Both GAA and GAG specify glutamate, but no other amino acid
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Genetic code is Rosetta stone of life
4 During
transcription, one DNA strand, the template strand, provides a template for ordering the
sequence of nucleotides in an RNA transcript.
– the complementary RNA molecule is synthesized according to base-pairing
rules, except that uracil is the complementary base to adenine.
4 during translation, blocks of three nucleotides, codons, are decoded into a sequence of amino
acids.
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Transcription produces genetic messages in the form of
RNA
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Transcription produces genetic messages in form of RNA
4 Transcription
– transfer of genetic information from DNA to RNA
– occurs in cell nucleus
– RNA molecule is transcribed from DNA template
• in process that resembles replication of DNA strand
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Transcription produces genetic messages in form of RNA
4 Transcription
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– RNA polymerase
• separates the DNA strands at the appropriate point and bonds the RNA nucleotides as
they base-pair along the DNA template.
• like DNA polymerases, RNA polymerases can add nucleotides only to the 3’ end of the
growing polymer.
• Genes are read 3’->5’, creating a 5’->3’ RNA molecule.
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Transcription produces genetic messages in form of RNA
4 Transcription
– specific sequences of nucleotides along the DNA mark where gene transcription begins and
ends.
• RNA polymerase
– attaches and initiates transcription at the promotor
» “upstream” of the information contained in the gene, the transcription unit.
– terminator signals the end of transcription.
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The End of
Sections 10.1 - 10.9
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