General Biology (Bio107) Chapter 10 – Molecular Biology of the Gene

General Biology (Bio107)

Chapter 10 – Molecular Biologyof the Gene

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

3D Structure of DNA

• In April 1953, James Watson and Francis Crick shook the scientific world with an elegant double-helical model for the structure of deoxyribonucleic acid or DNA.

• Your genetic endowment is the DNA and its genetic information you inherited from your parents.

• Nucleic acids are unique in their ability to direct their own replication.

• To assure successful regeneration and reproduction using mitosis and meiosis, parental DNA has to be copied into two new strands of daughter DNA.

• Life relies on a process called DNA replication to achieve this.

Introduction


• Once T.H. Morgan’s group showed that genes are located on chromosomes, the two constituents of chromosomes - proteins and DNA - were the candidates for the genetic material.

• Until the 1940s, the great heterogeneity and specificity of function of proteins seemed to indicate that proteins were the genetic material.

• However, this was not consistent with experiments with microorganisms, like bacteria and viruses.

1. The search for genetic material lead to DNA


• The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928.

• He studied Streptococcus pneumoniae, a bacterium that causes pneumonia in mammals.– One strain, the R strain, was harmless.– The other strain, the S strain, was pathogenic.

• In an experiment Griffith mixed heat-killed S strain with live R strain bacteria and injected this into a mouse.

• The mouse died and he recovered the pathogenic strain from the mouse’s blood.


• Griffith called this phenomenon transformation, a change in genotype and phenotype due to the assimilation of a foreign substance (now known to be DNA) by a cell.


• For the next 14 years scientists tried to identify the transforming substance.

• Finally in 1944, Oswald Avery, Maclyn McCarty and Colin MacLeod announced that the transforming substance was DNA.

• 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.


• Further evidence that DNA was the genetic material was derived from studies that tracked the infection of bacteria by viruses.

• 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.

• Viruses that specifically attack bacteria are called bacteriophages or just phages.


• In 1952, Alfred Hershey & Martha Chase showed that DNA was the genetic material of the phage T2.

• The T2 phage, consisting almost entirely of DNA and protein, attacks Escherichia coli (E. coli), a common intestinal bacteria of mammals.

• This phage can quickly turn an E. coli cell into a T2-producing factory that releases phages when the cell ruptures.


• The famous Hershey and Chase experiment.• They radio-labeled protein (35S) and DNA (32P) of

T2 and then tracked which entered the E. coli cell during infection.– They grew one batch of T2 phage in the presence of

radioactive sulfur, marking the proteins but not DNA.– They grew another batch in the presence of radioactive

phosphorus, marking the DNA but not proteins.– They 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.


– The mixtures were spun in a centrifuge which separated the heavier bacterial cells in the pellet from lighter free phages and parts of phage in the liquid supernatant.

– They then tested the pellet and supernatant of the separate treatments for the presence of radioactivity.


• Hershey and Chase found that when the bacteria had been infected with T2 phages that contained radio-labeled proteins, most of the radioactivity was in the supernatant, not in the pellet.

• When they examined the bacterial cultures with T2 phage that had radio-labeled DNA, most of the radioactivity was in the pellet with the bacteria.

• Hershey and Chase concluded that the injected DNA of the phage provides the genetic information that makes the infected cells produce new viral DNA and proteins, which assemble into new viruses.


• By 1947, Erwin Chargaff had developed a series of rules based on a survey of DNA composition in organisms.– He already knew that DNA was a polymer of

nucleotides consisting of a nitrogenous base, deoxyribose, and a phosphate group.

– The bases could be adenine (A), thymine (T), guanine (G), or cytosine (C).

• Chargaff noted that even though the DNA composition varies from species to species, the four bases are found in characteristic, but not necessarily equal, ratios in different species.


• He also found a peculiar regularity in the ratios of nucleotide bases which are known as Chargaff’s rule.

• The number of adenines was approximately equal to the number of thymines (%T = %A).

• The number of guanines was approximately equal to the number of cytosines (%G = %C).– Human DNA is 30.9% adenine, 29.4% thymine,

19.9% guanine and 19.8% cytosine.


• By the beginnings of the 1950’s, the race was on to move from the structure of a single DNA strand to the three-dimensional structure of DNA.

– Among the scientists working on the problem were Linus Pauling, in California, and Maurice Wilkins and Rosalind Franklin, in London.

2. Watson and Crick discovered the double helix by building models to conform to X-

ray data



• The phosphate group of one nucleotide is attached to the sugar of the next nucleotide in line.

• The result is a “backbone” of alternating phosphates and sugars, from which the bases project.

• Maurice Wilkins & Rosalind Franklin used X-ray crystallography to study the structure of DNA.– In this technique, X-rays are diffracted as they

passed through aligned fibers of purified DNA.– The diffraction pattern can be used to deduce

the three-dimensional shape of molecules.• James Watson learned

from their research that DNA was helical in shape and he deducedthe width of the helixand the spacing of bases.


• Watson and his colleague Francis Crick began to work on a model of DNA with two strands, the DNA double helix.

• Using molecular models made of wire, they first tried to place the sugar-phosphate chains on the inside.

• However, this did not fit the X-ray measurements and other information on the chemistry of DNA.


• The key breakthrough towards unraveling the 3D structure of DNA came when Watson put the sugar-phosphate chain on the outside and the nitrogen bases on the inside of the double helix.– The sugar-phosphate chains of each strand are

like the side ropes of a rope ladder.– Pairs of nitrogen bases, one from each strand,

form rungs.– The ladder forms a twist every ten bases.


• The nitrogenous bases are paired in specific combinations: adenine with thymine and guanine with cytosine.

• Pairing like nucleotides did not fit the uniform diameter indicated by the X-ray data.– A purine-purine pair would be too wide and a

pyrimidine-pyrimidine pairing would be too short.– Only a pyrimidine-

purine pairing would produce the 2-nm diameter indicated by the X-ray data.


Watson-Crick Base-pairing Rule

• In addition, Watson and Crick determined that chemical side groups off the nitrogen bases would form hydrogen bonds, connecting the two strands.

– Based on details of their structure, adenine would form two hydrogen bonds only with thymine and guanine would form three hydrogen bonds only with cytosine.

– This finding explained Chargaff’s rules.


• The base-pairing rules dictate the combinations of nitrogenous bases that form the “rungs” of DNA.

• However, this does not restrict the sequence of nucleotides along each DNA strand.

• The linear sequence of the four bases can be varied in countless ways.

• Each gene has a unique order of nitrogen bases.• In April 1953, Watson and Crick published a

succinct, one-page paper in Nature reporting their double helix model of DNA.


The DNA Molecule Polymer of nucleotides:

Adenine, thymine, cytosine, and guanine

Two polynucleotide strands form double helix associated with proteins

"Backbone" is deoxyribose-phosphate

Center contains the nitrogenous bases

• In a second paper Watson and Crick published their hypothesis for how DNA becomes copied within cells by a process called DNA replication.– Essentially, because each strand is

complementary to each other, each can form a template when separated.

– The order of bases on one strand can be used to add in complementary bases and therefore duplicate the pairs of bases exactly.

1. During DNA replication, base pairing enables existing DNA strands to serve as templates for new complimentary strands


• When a cell copies a DNA molecule, each parental strand serves as a template for ordering nucleotides into a new complimentary strand.– One at a time, nucleotides line up along the template

strand according to the base-pairing rules.– The nucleotides are linked to form new strands.


• Watson and Crick’s model, semiconservative replication, predicts that when a double helix replicates each of the daughter molecules will have one old strand and one newly made strand.

• Other competing models, the conservative model and the dispersive model, were also proposed.


• Experiments in the late 1950s by Matthew Meselson & Franklin Stahl supported the semiconservative model, proposed by Watson and Crick, over the other two models.– In their experiments, they labeled the

nucleotides of the old strands with a heavy isotope of nitrogen (15N) while any new nucleotides would be indicated by a lighter isotope (14N).

– Replicated strands could be separated by density in a centrifuge.

– Each model: the semi-conservative model, the conservative model, and the dispersive model, made specific predictions on the density of replicated DNA strands.



• The first replication in the 14N medium produced a band of hybrid (15N-14N) DNA, eliminating the conservative model.

• A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model.

Figure 8.3b

DNA Molecule

• The two strands are held together by hydrogen bonds between AT and CG

• Strands are antiparallel regarding 5’ and 3’ ends

DNA Replication

• Sustainability of life is guaranteed due copying of DNA by a process called DNA replication

“DNA characters (= nucleotides) are copied with an accuracy that rivals anything modern engineering can do.

They are just copied down to generations, with just enough occasional errors to introduce variety

(into the genetic material) …” R. Dawkins: River out of Eden

Figure 8.3a

Semi-conservative DNA Replication

Synthesis of New DNA

DNA Synthesis

• DNA replication starts at origin (‘ori” region)• First event is unwinding by helicase enzyme

(forms two replication forks)• DNA is copied by DNA polymerase

– Proceeds in the 5' 3' direction– Initiated by single-stranded RNA primer– Leading strand is synthesized continuously– Lagging strand is synthesized discontinuously – Many Okazaki fragments on lagging strand– RNA primers are removed and Okazaki fragments

joined by a DNA polymerase and DNA ligase

Replication Fork Events

Figure 8.5

DNA Synthesis at Replication Fork

The “Leading-Lagging Strand Problem”

• DNA polymerase reads parental DNA strand only in 3’ --> 5’ direction, it is only able to run with the replication fork along the leading strand

• On the lagging strand, DNA polymerase synthesizes in the opposite direction of the movement of the replication fork

• Multiple primers have to be synthesized on the lagging strand to generate multiple start sites for the DNA polymerase

• Many Okazaki fragments temporarily appear on the lagging strand

The “Leading-Lagging Strand Problem”

Ligase Action

• The enzyme Ligase formsa continuous daugther DNA strand by closing the tiny 3'-5' gaps betweenthe different daugther DNAfragments (= Okazaki fragments)

Summary of DNA Replication

• It is a fast process- the DNA polymerase adds about 50 nucleotides per second to growing DNA daughter strand!

• It is a very accurate process- only about one out of 1 million nucleotides, incorporated into the newly formed daughter strand is incorrectly build-in or wrongly paired during “un-repaired” DNA replication!- intrinsic 3’-exonuclease (“proof reading”) activity of DNA polymerase reduces this low error rate to one mistake per every 109 nucleotides added

Proof Reading Activity of DNA POL

Figure 8.6

Replication of Bacterial DNA

• Messenger RNA is transcribed from the template strand of a gene.

• 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.

DNA Transcription is the DNA-directed synthesis of RNA


• 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.

– The terminator signals the end of transcription.

• Bacteria have a single type of RNA polymerase that synthesizes all RNA molecules.

• In contrast, eukaryotes have three RNA polymerases (I, II, and III) in their nuclei.– RNA polymerase II is used for mRNA

synthesis.


DNA Transcription

• DNA sequence of a gene is transcribed to make RNA (mRNA, tRNA, and rRNA)

• Transcription begins when RNA polymerase binds to the promoter sequence located in front of gene

• Transcription is performed by RNA polymerase enzyme

• Elongation of transcription proceeds in 5' 3' direction

• Transcription stops when it reaches theterminator sequence

• In a eukaryotic cell, almost all transcription occurs in the nucleus and translation occurs mainly at ribosomes in the cytoplasm.

• In addition, before the primary transcript can leave the nucleus it is modified in various ways during RNA processing before the finished mRNA is exported to the cytoplasm.


Figure 8.7

Transcription

Organization of Typical Bacterial Gene

Sensestrand

Anti-Sensestrand

ATG

TAC

RNA Polymerase

• Adds new nucleotides to growing RNA strand during DNA transcription

• Synthesizes RNA in

5’ 3’ direction)• Requires Mg and Zn as

cofactors

• DNA transcriptioncan beseparatedinto threestages:1. Initiation, 2. Elongation, and3. Termination.


Initiation of DNA Transcription

• Starts with binding of the RNA polymerase (POL) - Sigma factor complex to promoter sequence of a gene

• Prokaryotic promoter sequence is located about 35 bp before the transcription start site- contains a 6 base sequence, usually TTGACA- contains a TATAAT or Pribnow box sequence about 10 bp before the transcription start site

• Bound RNA POL locally unwinds the DNA double helix of the gene to form a "transcription bubble"

Bacteria

• Activated transcription factors plus RNA polymerase II assemble and form the transcription-initiation complex, or transcriptome

• Transcriptomes are large and include many components:1. RNA polymerase I, II or III (= POL) - RNA POL II synthesizes mRNA - large multi-protein complex with about 500,000 Da - inhibited by mushroom poison alpha-amanitin; - RNA POL I and RNA POL III synthesize rRNA and tRNA2. TBP (= TATA box-binding protein) - binds to the TATA box of the promoter region3. Many Transcription factors, e.g. TFII F, TFII E, TFII H and TFII D - important for regulation of transcription

Initiation of Eukaryotic DNA Transcription

Figure 8.7

The Process of Transcription

Initiation

Elongation

• As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at time.

• The enzyme addsnucleotides to the3’ end of thegrowing strand.

• Behind the pointof RNA synthesis,the double helixre-forms and theRNA moleculepeels away.


Figure 8.7

ANIMATION Transcription: Overview

ANIMATION Transcription: Process

The Process of Transcription

Elongation

Termination

Termination of DNA Transcription

• Terminator sequence on gene sets the stop signal for DNA transcription

• Prokaryotic terminators often contain two types of stop signals:1. Poly-uridine-(poly-U) based termination - consists of 6 uridine residues (UUUUUU) following a hairpin structure - no protein factors required2. Rho-dependent termination - lacks a poly-U region, and often the hairpin loop; - requires the protein factor "rho"

Organization of Eukaryotic Gene

• Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm.

• At the 5’ end of the pre-mRNA molecule, a modified form of guanine is added, the 5’ cap.– This helps protect mRNA from hydrolytic

enzymes.– It also functions as an “attach here” signal for

ribosomes.

Eukaryotic cells modify mRNA after transcription


• At the 3’ end, an enzyme adds 50 to 250 adenine nucleotides, the poly(A) tail.– In addition to inhibiting hydrolysis and

facilitating ribosome attachment, the poly(A) tail also seems to facilitate the export of mRNA from the nucleus.

• The mRNA molecule also includes nontranslated leader and trailer segments.


• The most remarkable stage of RNA processing occurs during the removal of a large portion of the RNA molecule during RNA splicing.

• Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides.– Noncoding segments, introns, lie between

coding regions.– The final mRNA transcript includes coding

regions, exons, that are translated into amino acid sequences, plus the leader and trailer sequences.


Splicing of mRNA in Eukaryotes


• RNA splicing removes introns and joins exons to create an mRNA molecule with a continuous coding sequence.

mRNA Splicing

• Splicing = cutting out of the non-coding intron regions from nascent mRNA

• Two different splicing mechanisms are known:

1. protein-independent self-splicing - requires catalytically active snRNA

2. spliceosome-dependent splicing - requires several RNA and protein components, e.g. U1, U2, U4, U5, U6 snRNA and hnRNPs - most introns are spliced by this mechanism

• RNA splicing appears to have several functions.– First, at least some introns contain sequences

that control gene activity in some way.– Splicing itself may regulate the passage of

mRNA from the nucleus to the cytoplasm.– One clear benefit of split genes is to enable a

one gene to encode for more than one polypeptide.

• Alternative RNA splicing gives rise to two or more different polypeptides, depending on which segments are treated as exons.– Early results of the Human Genome Project

indicate that this phenomenon may be common in humans.


• Split genes may also facilitate the evolution of new proteins.

• Proteins often have amodular architecturewith discrete structuraland functional regionscalled domains.

• In many cases, different exons code for different domains of a protein.


RNA Processing Steps

• 3 major mRNA processing steps have been identified in cells1. CAPing of the 5’-end of the pre-pro-mRNA - covalent attachment of a chemical group(m7GpppNmpN) - done by capping enzyme and methyl transferase

2. Attachment of Poly-A tail to 3’ end of mRNA

3. Splicing of introns (eukaryotic mRNA only) - ATP-dep. polymerization of >200 adenine residues to the 3’-end of nascent mRNA molecule - catalyzed by Poly-A polymerase (PAP)

Poisons & Inhibitors of DNA Transcription

Protein Translation

• Nucleotide sequence of mRNA is translated at the ribosome in codons (three nucleotides) steps

• Translation of mRNA begins at the start codon: AUG

• Translation ends at noncoding codons: UAA, UAG, UGA

• During transcription, one DNA strand, the template strand, provides a template for ordering the sequence of nucleotides in an RNA transcript.

• During translation, blocks of three nucleotides, codons, are decoded into a linear sequence of linked amino acids.


• During translation, each type of tRNA links a mRNA codon with the appropriate amino acid.

• Each tRNA arriving at the ribosome carries a specific amino acid at one end and has a specific nucleotide triplet, an anticodon, at the other.

• The anticodon base-pairs with a complementary codon on mRNA.– If the codon on mRNA is UUU, a tRNA with an

AAA anticodon and carrying phenyalanine will bind to it.

• Codon by codon, tRNAs deposit amino acids in the prescribed order and the ribosome joins them into a polypeptide chain.


• A tRNA molecule consists of a strand of about 80 nucleotides that folds back on itself to form a three-dimensional structure.– It includes a loop containing the anticodon and

an attachment site at the 3’ end for an amino acid.


Transfer RNA (tRNA)

• Has complex 3-dimensional structure with two major interaction domains

1. Amino acid attachment site - site where corresponding amino acid is covalently attached to tRNA

2. Anti-codon site - Watson-Crick base pairs with complementary codon on mRNA strand

• If each anticodon had to be a perfect match to each codon, we would expect to find 61 types of tRNA, but the actual number is about 45.

• The anticodons of some tRNAs recognize more than one codon.

• This is possible because the rules for base pairing between the third base of the codon and anticodon are relaxed (called wobble).– At the wobble position, U on the anticodon can

bind with A or G in the third position of a codon.

– Some tRNA anticodons include a modified form of adenine, inosine, which can hydrogen bond with U, C, or A on the codon.


• If the genetic code consisted of a single nucleotide or even pairs of nucleotides per amino acid, there would not be enough combinations (4 and 16 respectively) to code for all 20 amino acids.

• Triplets of nucleotide bases are the smallest units of uniform length that can code for all the amino acids.

• In the triplet code, three consecutive bases specify an amino acid, creating 43 (64) possible code words.

• The genetic instructions for a polypeptide chain are written in DNA as a series of three-nucleotide words.

In the genetic code, nucleotide triplets specify amino acids


• During protein translation at the ribosome, the codons are read in the 5’->3’ direction along the mRNA.

• Each codon specifies which one of the 20 amino acids will be incorporated at the corresponding position along a polypeptide.

• Because codons are base triplets, the number of nucleotides making up a genetic message must be three times the number of amino acids making up the protein product.– It would take at least 300 nucleotides to code for

a polypeptide that is 100 amino acids long.


• Ribosomes facilitate the specific coupling of the tRNA anticodons with mRNA codons.– Each ribosome has a large and a small subunit.– These are composed of proteins and ribosomal RNA

(rRNA), the most abundant RNA in the cell.


• Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules.– The P site holds the tRNA carrying the

growing polypeptide chain.– The A site carries the tRNA with the next

amino acid.– Discharged tRNAs leave the ribosome at the E

site.


Protein Translation

• Requires the following cell components:

1. mature mRNA2. amino-acyl tRNA

3. large and small ribosome units

4. rRNA

5. protein factors, e.g. IFs, EFs and RFs

6. Cell energy, in form of ATP, GTP

Ribosomal RNAs (rRNA)

• Structural and functional part of the ribosomes.

• Three forms of rRNA each with different length and function exist:1. Prokaryotic organisms: 5S-rRNA; 16S-rRNA & 23S-rRNA 2. Eukaryotic organisms: 7S-rRNA; 18S-rRNA & 28S-rRNA

• Synthesized (= transcribed) in high amount in the nucleolus.

Figure 8.9

The Process of Translation

+ Initiation factors (IFs)+ GTP

1. Initiation

• Elongation consists of a series of three stepcycles as each amino acid is added to the proceeding one.

• During codon recognition, an elongation factor assists hydrogen bonding between the mRNA codon under the A site with the corresonding anticodon of tRNA carrying the appropriateamino acid.– This step requires the hydrolysis of two GTP.


2. Transamination

• During peptide bond formation (“transpeptidylation” or “transamination”), an rRNA molecule catalyzes the formation of a peptide bond between the polypeptide in the P site with the new amino acid in the A site.

• This step separates the tRNA at the P site from the growing polypeptide chain and transfers the chain, now one amino acid longer, to the tRNA at the A site.


3. Translocation & Elongation

+ Elongation factors (EFs)+ GTP

• During translocation, the ribosome moves the tRNA with the attached polypeptide from the A site to the P site.– Because the anticodon remains bonded to the

mRNA codon, the mRNA moves along with it.– The next codon is now available at the A site.– The tRNA that had been in the P site is moved

to the E site and then leaves the ribosome.– Translocation is fueled by the hydrolysis of

GTP.– Effectively, translocation ensures that the

mRNA is “read” 5’ -> 3’ codon by codon.


• The three steps of elongation continue codon by codon to add amino acids until the polypeptide chain is completed.


4. Polypeptide Chain Elongation


RF

5. Termination

+ Releasing Factor (RF)+ Water


The Genetic Code

• 61 sense codons on mRNA encode the 20 amino acids

• The genetic code is degenerate

• tRNA carries the complementary anticodon

• The task of matching each codon to its amino acid counterpart began in the early 1960s.

• Marshall Nirenberg determined the first match, that UUU coded for the amino acid phenylalanine.– He created an artificial mRNA molecule entirely

of uracil and 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.

• Other more elaborate techniques were required to decode mixed triplets such a AUA and CGA.


The Genetic Code

The genetic code of lifeis redundant; i.e. more than one codon codes for one amino acid

• The genetic code is nearly universal, shared by organisms from the simplest bacteria to the most complex plants and animals.

• This enables genetic engineering.• In laboratory experiments,

genes can be transcribed and translated after they are transplanted from one species to another.– This tobacco plant is expressing

a transpired firefly gene.

The genetic code must have evolved very early in the history of life

• The near universality of the genetic code must have been operating very early in the history of life.

• A shared genetic vocabulary is a reminder of the kinship that bonds all life on Earth.


• Although bacteria and eukaryotes carry out transcription and translation in very similar ways, they do have differences in cellular machinery andin details of the processes.– Eukaryotic RNA polymerases differ from those of

prokaryotes and require transcription factors.– They differ in how transcription is terminated.– Their ribosomes are also different.

Comparing protein synthesis in prokaryotes and eukaryotes: a review


• In one big differences, prokaryotes can transcribe and translate the same gene simultaneously.

• The new protein quickly diffuses to its operating site.


• In eukaryotes, the nuclear envelope segregates transcription from translation.

• In addition, extensive RNA processing is inserted between these processes.– This provides additional steps whose

regulation helps coordinate the elaborate activities of a eukaryotic cell.

• In addition, eukaryotic cells have complicated mechanisms for targeting proteins to the appropriate organelle.


• Transcription, RNA processing, and translation are the processes that link DNA sequences to the synthesis of a specific polypeptide chain.


Regulation of Protein Translation• Gene expression is also regulated at the protein translation level at

the ribosome• Regulation of protein translation happens via:

1. Control by protein phosphorylation of initiation factors - phosphorylation of eIF-2 by heme-controlled inhibitor (HCI) blocks exchange of the bound GDP for GTP

2. Control by Interferons (IFs) - IFs are cellular signaling molecules that activate RNA-dependent protein kinases (= PKRs) - PKRs phosphorylate and inactivate eIF-2 therefore preventing initiation of protein translation - most IFs are released from white blood cells after viral attack - 3 different classes of interferons are known

3. Control by post-translational protein modification 1. Glycosylation (= attachment of sugar residues to proteins) 2. Isoprenylation/Acylation (= attachment of isoprenoid or fatty acids) 3. Ubiquitination & Proteolytic Digest (Proteasome, Apoptosis --> Caspases)

Figure 8.12

ANIMATION Operons: Overview

Regulation in Bacteria: Operon Model

Polycistronic mRNA

Figure 8.12

Induction – Lac Operon

1. Lactose absent

No Transcription!

Figure 8.12

Induction – Lac Operon

2. Lactose present

DNA Transcription

Mutation

• A change in the genetic material.• Mutations change the DNA sequence and

therefore the genetic information.• Mutations may be neutral, beneficial, or harmful• Mutagen: Agent that causes mutations• Spontaneous mutations: Occur in the absence

of a mutagen

• Base substitution (point mutation)

• Missense mutation

Types of Mutations

• Change in one base

• Result in change in amino acid

Figure 8.17a, b

• For example, sickle-cell disease is caused by a mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin. – A change in a single nucleotide from T to A in

the DNA template leads to an abnormal protein.


• Nonsense mutation • Results in a nonsense codon

Figure 8.17a, c

• Frameshift mutation • Insertion or deletion of one or more nucleotide pairs

Figure 8.17a, d

• Spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated genes

• Mutagens increase to 10–5 or 10–3 per replicated gene

• Mutation rate vastly responsible for “pace of evolutionary change” of life forms

ANIMATION Mutations: Types

The Frequency of Mutation

Figure 8.19a

Chemical Mutagens

Figure 8.19b

ANIMATION Mutagens

Chemical Mutagens

Chemical mutagens & Carcinogens•the molecules shown below act as mutagens and can cause cancers•cancer-causing chemicals are called carcinogens•all chemicals carcinogens are mutagens, i.e. are able to interact with DNA

Require prior “metabolic activation” by (Cyt P450) liver enzymes

Radiation

• Ionizing radiation (X rays and gamma rays) causes the formation of ions that can react with nucleotides and the deoxyribose-phosphate backbone- often cause DNA double strand breaks (DSBs) with dangerous consequences

Figure 8.20

Radiation

• UV radiation causes thymine dimer formationof neighboringthymines (T) inDNA.

Mutations & DNA Repair

• Photolyases separate thymine dimers• Nucleotide excision repair (NER)

ANIMATION Mutations: Repair

• In mismatch repair, special enzymes fix incorrectly paired nucleotides.– A hereditary defect in

one of these enzymesis associated with a form of colon cancer.

• In nucleotide excision repair, a nuclease cuts out a segment of a damaged strand.– The gap is filled in by

DNA polymerase and ligase.


• The importance of proper function of repair enzymes is clear from the inherited disorder xeroderma pigmentosum.– These individuals are hypersensitive to

sunlight.– In particular, ultraviolet light can produce

thymine dimers between adjacent thymine nucleotides.

– This buckles the DNA double helix and interferes with DNA replication.

– In individuals with this disorder, mutations in their skin cells are left uncorrected and cause skin cancer.


Ionizing radiation (X-ray)Oxygen radicals (ROS)Bleomycin, CisplatinIncorrect base

incorporation during DNA replication

DNA POL III

UVirradiation

MutagenicChemicals,

ROS

Stop ofDNA Polymerase

T- dimerformation

Base (N) modification

((O6- ethyl-G, 8-OxoG)++

Mis-matched pairing

Double-strandDouble-strandbreak (DSB)break (DSB)

mutmutHLS,HLS,OGG1OGG1

UvrUvrABCABCXPCXPC

KuKuheterodimerheterodimer

NucleotideNucleotideExcision repairExcision repair

(NER)(NER)

DNA POLDNA POLProof readingProof reading

MismatchMismatchrepairrepair

HomologousHomologousrecombination (HR)recombination (HR)

ororNH end joiningNH end joining

DNA POLDNA POL3’-exo-3’-exo-nucleasenuclease

Graphics©E.Schmid/2002

Ames Test for Chemical Carcinogens

Figure 8.22

Ames Test for Chemical Carcinogens

• The ends of eukaryotic chromosomal DNA molecules, the telomeres, have special nucleotide sequences.– In human telomeres, this sequence is typically

TTAGGG, repeated between 100 and 1,000 times.

• Telomeres protect genes from being eroded through multiple rounds of DNA replication.


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General Biology (Bio107) Chapter 10 – Molecular Biology of the Gene