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Molecular Biology of the Gene Bio 103 Lecture Dr. Largen 2

4Finding the genetic material 4Structure of the genetic material 4DNA replication 3 4

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 5

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 6

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. 7

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. 8

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 9

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 10 11

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. 12

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. 13

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. 14 15

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 17

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 21 22 23

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 25 26

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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 32 33 34

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 35 36 37

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 38 39

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 41

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 44 45 46

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. 47

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. 48 49

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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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 52

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 53

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 54

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 55 56 57

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. 58

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. 59

DNA genotype expressed as proteins 4 Flow

of genetic information in a eukaryotic cell:

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– transcription • RNA – translation » protein 60 61

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 63

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 65 66

Genetic code is Rosetta stone of life

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Genetic code is Rosetta stone of life

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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 69

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 70

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. 73 74

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 76

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. 77

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. 78 79 80

The End of Sections 10.1 - 10.9

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