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What We Know

DNA Replication

Chapter 25

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DNA Structure

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DNA Structure and Replication

• Wilkins and Franklin studied the structure of DNA using x-ray diffraction photography

• Watson and Crick discovered DNAs structure

• In the mid-1900s, scientists knew that chromosomes, made up of DNA (deoxyribonucleic acid) and proteins, contained genetic information.

• However, they did not know whether the DNA or the proteins was the actual genetic material.

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• Various researcher's showed that DNA was the genetic material when they performed an experiment with a T2 virus.

• By using different radioactively labeled components, they demonstrated that only the virus DNA entered a bacterium to take over the cell and produce new viruses.

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Viral DNA is labeled

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Viral capsid is labeled

Structure of DNA• The structure of DNA was determined by James Watson and

Francis Crick in the early 1950s.• DNA is made up of nucleotides

Nucleotides • Phosphate• A sugar • A nitrogen-containing base.

DNA• Pentose sugar;• Four different bases: adenine (A), thymine (T), guanine (G), and

cytosine (C).

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One pair of bases

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• Watson and Crick showed that DNA is a double helix in which A is paired with T and G is paired with C.

• This is called complementary base pairing because a purine is always paired with a pyrimidine.

• Purine=

• Pyrimidine=

• When the DNA double helix unwinds, it resembles a ladder.

• The sides of the ladder are the sugar-phosphate backbones

• The rungs of the ladder are the complementary paired bases.

• The two DNA strands are anti-parallel – they run in opposite directions (3’ and 5’).

Chargaff's rules state that DNA from any cell of all organisms should have a 1:1 ratio (base Pair Rule) of pyrimidine and purine bases and, more specifically, that the amount of guanine is equal to cytosine and the amount of adenine is equal to thymine. This pattern is found in both strands of the DNA.

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DNA double helix

Replication of DNA• DNA replication occurs during chromosome

duplication• an exact copy of the DNA is produced with the aid

of DNA polymerase.

1. Hydrogen bonds between bases break and enzymes “unzip” the molecule.

2. Each old strand of nucleotides serves as a template for each new strand.

3. New nucleotides move into complementary positions are joined by DNA polymerase.

• The process is semiconservative because each new double helix is composed of an old strand of nucleotides from the parent molecule and one newly-formed strand.

• Some cancer treatments are aimed at stopping DNA replication in rapidly-dividing cancer cells.

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Overview of DNA replication

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Ladder configuration and DNA replication

DNA Replication

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RNA (ribonucleic acid)• single-stranded nucleic acid in which A pairs with U

(uracil) while G pairs with C.

Three types of RNA are involved in gene expression: • messenger RNA (mRNA) carries genetic information

to the ribosomes

• ribosomal RNA (rRNA) is found in the ribosomes

• transfer RNA (tRNA) transfers amino acids to the ribosomes, where the protein product is synthesized.

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Structure of RNA

Ribose

tRNA

rRNA• Made in the nucleolus, proteins join it to make the two

subunits of the ribosomes.

• Two processes are involved in the synthesis of proteins in the cell:

• Transcription makes an RNA molecule complementary to a portion of DNA.

• Translation occurs when the sequence of bases of mRNA directs the sequence of amino acids in a polypeptide.

Protein Synthesis

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The Genetic Code• DNA specifies the synthesis of proteins because

it contains a triplet code: every three bases stand for one amino acid.

• Each three-letter unit of an mRNA molecule is called a codon.

• Most amino acids have more than one codon; there are 20 amino acids with a possible 64 different triplets.

• The code is nearly universal among living organisms.

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Messenger RNA codons

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Central Concept• The central concept of genetics involves the DNA-

to-protein sequence involving transcription and translation.

• DNA has a sequence of bases that is transcribed into a sequence of bases in mRNA.

• Every three bases is a codon that stands for a particular amino acid.

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Overview of gene expression

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Transcription• During transcription in the nucleus, a segment

of DNA unwinds and unzips, and the DNA serves as a template for mRNA formation.

• RNA polymerase joins the RNA nucleotides so that the codons in mRNA are complementary to the triplet code in DNA.

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Transcription and mRNA synthesis

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Processing of mRNA• DNA contains exons and introns. • Before mRNA leaves the nucleus, it is

processed and the introns are excised so that only the exons are expressed.

• The splicing of mRNA is done by ribozymes, organic catalysts composed of RNA, not protein.

• Primary mRNA is processed into mature mRNA.

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Function of introns

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Translation• Translation is the second step by which gene

expression leads to protein synthesis.• During translation, the sequence of codons in

mRNA specifies the order of amino acids in a protein.

• Translation requires several enzymes and two other types of RNA: transfer RNA and ribosomal RNA.

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Transfer RNA

• During translation, transfer RNA (tRNA) molecules attach to their own particular amino acid and travel to a ribosome.

• Through complementary base pairing between anticodons of tRNA and codons of mRNA, the sequence of tRNAs and their amino acids form the sequence of the polypeptide.

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Transfer RNA: amino acid carrier

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Ribosomal RNA• Ribosomal RNA, also called structural RNA, is made

in the nucleolus.

• Proteins made in the cytoplasm move into the nucleus and join with ribosomal RNA to form the subunits of ribosomes.

• A large subunit and small subunit of a ribosome leave the nucleus and join in the cytoplasm to form a ribosome just prior to protein synthesis.

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• A ribosome has a binding site for mRNA as well as binding sites for two tRNA molecules at a time.

• As the ribosome moves down the mRNA molecule, new tRNAs arrive, and a polypeptide forms and grows longer.

• Translation terminates once the polypeptide is fully formed; the ribosome separates into two subunits and falls off the mRNA.

• Several ribosomes may attach and translate the same mRNA, therefore the name polyribosome.

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Polyribosome structure and function

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Translation Requires Three Steps

• During translation, the codons of an mRNA base-pair with tRNA anticodons.

• Protein translation requires these steps:1) Chain initiation2) Chain elongation3) Chain termination.• Enzymes are required for each step, and the

first two steps require energy.

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Chain Initiation

• During chain initiation, a small ribosomal subunit, the mRNA, an initiator tRNA, and a large ribosomal unit bind together.

• First, a small ribosomal subunit attaches to the mRNA near the start codon.

• The anticodon of tRNA, called the initiator RNA, pairs with this codon.

• Then the large ribosomal subunit joins.

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Initiation

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Chain Elongation• During chain elongation, the initiator tRNA

passes its amino acid to a tRNA-amino acid complex that has come to the second binding site.

• The ribosome moves forward and the tRNA at the second binding site is now at the first site, a sequence called translocation.

• The previous tRNA leaves the ribosome and picks up another amino acid before returning.

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Elongation

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Chain Termination• Chain termination occurs when a stop-codon

sequence is reached. • The polypeptide is enzymatically cleaved from

the last tRNA by a release factor, and the ribosome falls away from the mRNA molecule.

• A newly synthesized polypeptide may function along or become part of a protein.

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Termination

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Review of Gene Expression• DNA in the nucleus contains a triplet code;

each group of three bases stands for one amino acid.

• During transcription, an mRNA copy of the DNA template is made.

• The mRNA is processed before leaving the nucleus.

• The mRNA joins with a ribosome, where tRNA carries the amino acids into position during translation.

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Gene expression

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Control of Gene Expression

• The lac operon model explains how one regulator gene controls the transcription of several structural genes — genes that code for proteins.

• The promoter is a short sequence of DNA where RNA polymerase first attaches when a gene is to be transcribed.

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• The operator is a short sequence of DNA where the repressor protein binds to the operator and prevents RNA polymerase from attaching to another portion of DNA called the promoter.

• Transcription does not occur until lactose binds to the repressor preventing the repressor from binding to the operator.

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• Now RNA polymerase binds to the operator and brings about transcription of the genes that code for enzymes necessary to lactose metabolism.

• Structural genes code for enzymes of a metabolic pathway that are transcribed as a unit.

• A regulator gene codes for a repressor that can bind to the operator and switch off the operon; therefore, a regulator gene regulates the activity of structural genes.

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The lac operon

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Control of Gene Expression in Eukaryotes

• In eukaryotes, cells differ in which genes are being expressed.

• Levels of control in eukaryotes include: • transcriptional control,

• posttranscriptional control, • translational control, and • posttranslational control.

• The first two methods occur in the nucleus; the second two, in the cytoplasm.

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Eukaryotic control of gene expression

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Transcriptional Control in Eukaryotes

• Rarely are there operons in eukaryotic cells.• Instead, transcriptional control in eukaryotes

involves:1) The organization of the chromatin, and2) Regulator proteins called transcription

factors.

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Activated Chromatin• The existence of chromosome puffs in developing

eggs of many vertebrates suggests that DNA must decondense in order for transcription to occur.

• The chromosomes within many vertebrate egg cells are called lampbrush chromosomes because they have many decondensed loops; here mRNA is synthesized in great quantity.

• This form of transcriptional control is useful when the gene product is tRNA or rRNA.

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Lampbrush chromosomes

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Transcription Factors• Transcription factors regulate transcription of

DNA in eukaryotes. • Signals received from inside and outside the

cell turn on particular transcription factors. • Activation probably occurs when the

transcription factors are phosphorylated by a kinase.

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Gene Mutations

• A gene mutation is a change in the sequence of bases within a gene.

• Frameshift Mutations • Frameshift mutations involve the addition or

removal of a base during the formation of mRNA; these change the genetic message by shifting the “reading frame.”

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Point Mutations

• The change of just one nucleotide causing a codon change can cause the wrong amino acid to be inserted in a polypeptide; this is a point mutation.

• In a silent mutation, the change in the codon results in the same amino acid.

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• If a codon is changed to a stop codon, the resulting protein may be too short to function; this is a nonsense mutation.

• If a point mutation involves the substitution of a different amino acid, the result may be a protein that cannot reach its final shape; this is a missense mutation.

• An example is Hbs which causes sickle-cell disease.

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Sickle-cell disease in humans

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Cause and Repair of Mutations• Mutations can be spontaneous or caused by

environmental influences called mutagens. • Mutagens include radiation (X-rays, UV

radiation), and organic chemicals (in cigarette smoke and pesticides).

• DNA polymerase proofreads the new strand against the old strand and detects mismatched pairs, reducing mistakes to one in a billion nucleotide pairs replicated.

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Transposons: Jumping Genes

• Transposons are specific DNA sequences that move from place to place within and between chromosomes.

• These so-called jumping genes can cause a mutation to occur by altering gene expression.

• It is likely all organisms, including humans, have transposons.

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Cancer: A Failure of Genetic Control

• Cancer is a genetic disorder resulting in a tumor, an abnormal mass of cells.

• Carcinogenesis, the development of cancer, is a gradual process.

• Cancer cells lack differentiation, form tumors, undergo angiogenesis and metastasize.

• Cancer cells fail to undergo apoptosis, or programmed cell death.

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Cancer cells

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• Angiogenesis is the formation of new blood vessels to bring additional nutrients and oxygen to a tumor; cancer cells stimulate angiogenesis.

• Metastasis is invasion of other tissues by establishment of tumors at new sites.

• A patient’s prognosis is dependent on the degree to which the cancer has progressed; early diagnosis and treatment is critical to survival.

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Origin of Cancer• Mutations in at least four classes of genes are

associated with the development of cancer.• 1) The nucleus has a DNA repair system but

mutations in genes for repair enzymes can contribute to cancer.

• 2) Mutations in genes that code for proteins regulating structure of chromatin can promote cancer.

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• 3) Proto-oncogenes are normal genes that stimulate the cell cycle and tumor-suppressor genes inhibit the cell cycle; mutations can prevent normal regulation of the cell cycle.

• 4) Telomeres are DNA segments at the ends of chromosomes that normally get shorter and signal an end to cell division; cancer cells have an enzyme that keeps telomeres long.

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Regulation of Cell Division• Proto-oncogenes are part of a stimulatory

pathway that extends from membrane to nucleus.

• Tumor-suppressor genes are part of an inhibitory pathway extending from the plasma membrane to the nucleus.

• The balance between stimulatory signals and inhibitory signals determines whether proto-oncogenes or tumor-suppressor genes are active.

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• Plasma membrane receptors can receive growth stimulatory factors and growth inhibitory factors.

• Cytoplasmic proteins can therefore be turned on or off and in turn either stimulate or inhibit certain genes in the nucleus.

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Oncogenes• Proto-oncogenes can undergo mutations to

become cancer-causing oncogenes. • An oncogene may code for a faulty receptor in

the stimulatory pathway.• Or an oncogene may produce either an

abnormal protein product or abnormally high levels of a normal protein product that stimulates the cell cycle to begin or to go to completion; both lead to uncontrolled growth.

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• About 100 oncogenes have been discovered that cause increased growth and lead to tumors.

• Alteration of a single nucleotide pair can convert a normal rasK proto-oncogene to an oncogene implicated in lung, colon, and pancreatic cancer.

• The rasN oncogene is associated with leukemia and lymphoma.

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Tumor-Suppressor Genes

• Tumor-suppressor genes ordinarily suppress the cell cycle; when they mutate they stop suppressing the cell cycle and it can occur nonstop.

• RB tumor-suppressor gene malfunctions are implicated in cancers of the breast, prostate, bladder, and small-cell lung carcinoma.

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• Another major tumor-suppressor gene is p53, a gene that is more frequently mutated in human cancers than any other known gene.

• The p53 protein acts as a transcription factor and as such is involved in turning on the expression of genes whose products are cell cycle inhibitors.

• P53 can also stimulate apoptosis.

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Causes of cancer

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Chapter Summary

• Since DNA is the genetic material, its structure and functions constitute the molecular basis of inheritance.

• Because the DNA molecule is able to replicate, genetic information can be passed from one cell generation to the next.

• DNA codes for the synthesis of proteins; this process also involves RNA.

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• In prokaryotes, regulator genes control the activity and expression of other genes.

• In eukaryotes, the control of gene expression occurs at all stages, from transcription to the activity of proteins.

• Gene mutations vary; some have little effect but some have a dramatic effect.

• Loss of genetic control over genes involved in cell growth and/or cell division cause cancer.