12.1 The Genetic Material
1. Early researchers knew that the genetic material must be:
a. Able to store information used to control both the development and the metabolic activities of cells;
b. Stable so it can be replicated accurately during cell division and be transmitted for generations; and,
c. Able to undergo mutations providing the genetic variability required for evolution.
A. Transformation of Bacteria
1. Bacteriologist Frederick Griffith (1931) experimented with Streptococcus pneumoniae (a pneumococcus that causes pneumonia in mammals).
2. Mice were injected with two strains of pneumococcus: an encapsulated (S) strain and a non-encapsulated (R) strain.
a. The S strain is virulent (the mice died); it has a mucous capsule and forms “shiny” colonies.
b. The R strain is not virulent (the mice lived); it has no capsule and forms “dull” colonies.
3. In an effort to determine if the capsule alone was responsible for the virulence of the S strain, he injected mice with heat-killed S strain bacteria; the mice lived.
4. Finally, he injected mice with a mixture of heat-killed S strain and live R strain bacteria.
a. The mice died; living S strain pneumococcus was recovered from their bodies.
b. Griffith concluded that some substance necessary for synthesis of the capsule—and therefore for virulence—must pass from dead S strain bacteria to living R strain bacteria so the R strain were transformed.
c. This change in phenotype of the R strain must be due to a change in the bacterial genotype, suggesting that the transforming substance passed from S strain to R strain.
B. DNA: The Transforming Substance
1. Oswald Avery et al. (1944) reported that the transforming substance was DNA.
2. In the early twentieth century, it was shown that nucleic acids contain four types of nucleotides.
a. DNA was composed of repeating units, each of which always had just one of each of four different nucleotides (a nitrogenous base, a phosphate, and a pentose).
b. In this model, DNA could not vary between species and therefore could not be the genetic material; therefore some other protein component was expected to be the genetic material.
3. Purified DNA is capable of bringing about the transformation. Evidence:
a. DNA from S strain pneumococcus causes R strain bacteria to be transformed.
b. Digestion of the transforming substance with enzyme that digests DNA prevents transformation.
c. The molecular weight of the transforming substance is great enough for
some genetic variability.
d. Enzymes that degrade proteins cannot prevent transformation, nor can enzymes that digest RNA.
4. Avery’s experimental results demonstrated DNA is genetic material and DNA controls biosynthetic properties of a cell.
C. Transformation of Organisms Today
1. Transformation experiments today are important especially in biotechnology labs.
2. Transformation of organisms is being used in commercial products.
3. In order to illustrate that transferring genes was possible from one organism to another, scientists used a green fluorescent protein from jellyfish and transferred it to other organisms. The result was that these organisms glowed in the dark.
4. Mammalian genes have the ability to function in other species: bacteria, invertebrates, plants.
D. The Structure of DNA
1. Erwin Chargaff (1940s) analyzed the base content of DNA.
2. It was known DNA contained four different nucleotides:
a. Two with purine bases, adenine (A) and guanine (G); a purine is a type of nitrogen-containing base having a double-ring structure.
b. Two with pyrimidine bases, thymine (T) and cytosine (C); a pyrimidine is a type of nitrogen-containing base having a single-ring structure.
3. Results: DNA does have the variability necessary for the genetic material.
4. For a species, DNA has the constancy required of genetic material.
5. This constancy is given in Chargaff’s rules:
a. The amount of A, T, G, and C in DNA varies from species to species.
b. In each species, the amount of A = T and the amount of G = C (A +G = T +C).
6. The tetranucleotide hypothesis (proposing DNA was repeating units of one of four bases) was disproved: each species has its own constant base composition.
7. The variability is in base sequences is staggering; a human chromosome contains about 140 million base pairs.
8. Since any of the four possible nucleotides can be present at each nucleotide position, the total number of possible nucleotide sequences is 4140 x 106 = 4140,000,000.
9. Rosalind Franklin produced X-ray diffraction photographs.
10. Franklin’s work provided evidence that DNA had the following features:
a. DNA is a helix.
b. Some portion of the helix is repeated.
11. American James Watson joined with Francis H. C. Crick in England to work on the structure of DNA.
12. Watson and Crick received the Nobel Prize in 1962 for their model of DNA.
13. Using information generated by Chargaff and Franklin, Watson and Crick constructed a model of DNA as a double helix with sugar-phosphate groups on the outside, and paired bases on the inside.
14. Their model was consistent with both Chargaff’s rules and Franklin’s X-ray diffraction studies.
15. Complementary base pairing is the paired relationship between
purines and pyrimidines in DNA: A is hydrogen-bonded to T and G is hydrogen-bonded to C.
12.2 Replication of DNA
1. DNA replication is the process of copying a DNA molecule. Replication is semiconservative, with each strand of the original double helix (parental molecule) serving as a template (mold or model) for a new strand in a daughter molecule. This process consists of:
a. Unwinding: old strands of the parent DNA molecule are unwound as weak hydrogen bonds between the paired bases are “unzipped” and broken by the enzyme helicase.
b. Complementary base pairing: free nucleotides present in the nucleus bind with complementary bases on unzipped portions of the two strands of DNA; this process is catalyzed by DNA polymerase.
c. Joining: complementary nucleotides bond to each other to form new strands; each daughter DNA molecule contains an old strand and a new strand; this process is also catalyzed by DNA polymerase.
A. Aspects of DNA Replication (Biological Systems reading)
1. For complementary base pairing to occur, the DNA strands need to be antiparallel, as discovered by Watson and Crick.
2. One strand of DNA is 5’ at the top and the other strand is 3’ at the top of the strand.
3. During replication the DNA polymerase can only join to the free 3’ end of the previous nucleotide.
4. DNA polymerase cannot start the synthesis of a DNA chain, so an RNA polymerase lays out an RNA primer that is complementary to the replicated strand.
5. Now the DNA polymerase can join the DNA nucleotides to the 3’ end of the new strand.
6. The helicase enzyme unwinds the DNA and one strand (called the leading new strand) can be copied in the direction of the replication fork.
7. The other strand of DNA is copied in the direction away from the fork, and replication begins again.
a. This new lagging strand is discontinuous and each segment is called an Okazaki fragment, after the scientist who discovered them.
8. Replication is only complete when RNA primers are removed.
9. During replication, DNA molecules gets smaller and smaller.
10. The end of eukaryotic DNA molecules have nucleotide sequences called telomeres.
a. Telomeres don’t code for proteins. They are repeats of short nucleotide sequences (i.e., TTAGGG).
11. Normal mammalian cells divide approximately 50 times and then stop. However in cancer cells the telomerase can be turned on and cancer cells then divide without limit.
B. Prokaryotic Versus Eukaryotic Replication
1. Prokaryotic DNA Replication
a. Bacteria have a single loop of DNA that must replicate before the cell divides.
b. Replication in prokaryotes may be bidirectional from one point of origin or in only one direction.
c. Replication only proceeds in one direction, from 5' to 3'.
d. Replication begins at a special site on a bacterial chromosome, called the origin of replication.
e. Bacterial cells can complete DNA replication in 40 minutes; eukaryotes take
2. Eukaryotic DNA Replication
a. Replication in eukaryotes starts at many points of origin and spreads with many replication bubbles—places where the DNA strands are separating and replication is occurring.
b. Replication forks are the V-shape ends of the replication bubbles; the sites of DNA replication.
c. Eukaryotes replicate their DNA at a slower rate—500 to 5,000 base pairs per minute.
d. Eukaryotes take hours to complete DNA replication.
3. Accuracy of Replication
a. A mismatched nucleotide may occur once per 100,000 base pairs, causing a pause in replication.
Proofreading is the removal of a mismatched nucleotide; DNA repair enzymes perform this proofreading function and reduce the error rate to one per billion base pairs.
12.3 The Genetic Code of Life
1. Sir Archibald Garrod (early 1900s) introduced the phrase inborn error of metabolism.
a. Garrod proposed that inherited defects could be caused by the lack of a particular enzyme.
b. Knowing that enzymes are proteins, Garrod suggested a link between genes and proteins.
2. George Beadle and Edward Tatum proposed the one gene, one enzyme hypothesis based on their study of red bread mold.
A. RNA Carries the Information
1. Like DNA, RNA (ribonucleic acid) is a polymer of nucleotides.
2. Unlike DNA, RNA is single-stranded, contains the sugar ribose, and the base uracil instead of thymine (in addition to cytosine, guanine, and adenine).
3. There are three major classes of RNA.
a. Messenger RNA (mRNA) takes a message from DNA in the nucleus to ribosomes in the cytoplasm.
b. Ribosomal RNA (rRNA) and proteins make up ribosomes where proteins are synthesized.
c. Transfer RNA (tRNA) transfers a particular amino acid to a ribosome.
B. The Genetic Code
DNA undergoes transcription to mRNA, which is translated to a protein.
DNA is a template for RNA formation during transcription.
Transcription is the first step in gene expression; it is the process whereby a DNA strand serves as a template for the formation of mRNA.
During translation, an mRNA transcript directs the sequence of amino acids in a polypeptide.
The process from DNA to protein is the central dogma of molecular biology.
The genetic code is a triplet code, comprised of three-base code words (e.g., AUG).
A codon consists of 3 nucleotide bases of DNA.
Four nucleotides based on 3-unit codons allows up to 64 different amino acids to the specified.
Finding the Genetic Code
a. Marshall Nirenberg and J. Heinrich Matthei (1961) found that an enzyme that could be used to construct synthetic RNA in a cell-free system; they showed the codon UUU coded for phenylalanine.
b. By translating just three nucleotides at a time, they assigned an amino acid to each of the RNA codons, and discovered important properties of the genetic code.
c. The code is degenerate: there are 64 triplets to code for 20 naturally occurring amino acids; this protects against potentially harmful mutations.
d. The genetic code is unambiguous; each triplet codon specifies one and only one amino acid.
e. The code has start and stop signals: there are one start codon and three stop codons.
10. The Code Is Universal
a. The few exceptions to universality of the genetic code suggest the code dates back to the very first organisms and that all organisms are related.
b. Once the code was established, changes would be disruptive.
12.4 First Step: Transcription
A. Messenger RNA Is Produced
A segment of the DNA helix unwinds and unzips.
Transcription begins when RNA polymerase attaches to a promoter on DNA. A promoter is a region of DNA which defines the start of the gene, the direction of transcription, and the strand to be transcribed.
As RNA polymerase (an enzyme that speeds formation of RNA from a DNA template) moves along the template strand of the DNA, complementary RNA nucleotides are paired with DNA nucleotides of the coding strand. The strand of DNA not being transcribed is called the noncoding strand.
RNA polymerase adds nucleotides to the 3'-end of the polymer under construction. Thus, RNA synthesis is in the 5’-to-3’ direction.
The RNA/DNA association is not as stable as the DNA double helix; therefore, only the newest portion of the RNA molecule associated with RNA polymerase is bound to DNA; the rest dangles off to the side.
Elongation of mRNA continues until RNA polymerase comes to a stop sequence.
The stop sequence causes RNA polymerase to stop transcribing DNA and to release the mRNA transcript.
Many RNA polymerase molecules work to produce mRNA from the same DNA region at the same time.
Cells produce thousands of copies of the same mRNA molecule and many copies of the same protein in a shorter period of time than if a single copy of RNA were used to direct protein synthesis.
B. RNA Molecules Undergo Processing
1. Newly formed pre-mRNA transcript is processed before leaving the nucleus.
2. Pre-mRNA transcript is the immediate product of transcription; it contains exons and introns.
3. The ends of the mRNA molecule are altered: a cap is put on the 5' end and a poly-A tail is put on the 3' end.
a. The “cap” is a modified guanine (G) where a ribosome attaches to begin translation.
b. The “poly-A tail” consists of a 150–200 adenine (A) nucleotide chain that facilitates transport of mRNA out of the nucleus and inhibits enzymatic degradation of mRNA.
4. Portions of the primary mRNA transcript, called introns, are removed.
a. An exon is a portion of the DNA code in the primary mRNA transcript eventually expressed in the final polypeptide product.
An intron is a non-coding segment of DNA removed by spliceosomes before the mRNA leaves the nucleus.
5. Ribozymes are RNAs with an enzymatic function restricted to removing introns from themselves.
a. RNA could have served as both genetic material and as the first enzymes in early life forms.
6. Spliceosomes are complexes that contain several kinds of ribonucleoproteins.
a. Spliceosomes cut the primary mRNA transcript and then rejoin adjacent exons.
7. Smaller nucleolar RNA (snoRNA) is present in the nucleolus, to assist in the processing of rRNA and tRNA molecules.
8. Introns give a cell the ability to decide which exons will go in a particular mRNA.
9. mRNA do not have all of the possible exons available from a DNA sequence. In one mRNA what is an exon could be an intron in another mRNA. This process is termed alternative mRNA splicing.
10. Some introns give rise to microRNAs (miRNA). miRNA regulate mRNA translation by bonding with mRNA through complementary base pairing and preventing translation from occurring.
11. Exon shuffling occurs when introns encourage crossing over during meiosis.
12.5 Second Step: Translation
1. Translation takes place in the cytoplasm of eukaryotic cells.
2. Translation is the second step by which gene expression leads to protein synthesis.
3. One language (nucleic acids) is translated into another language (protein).
A. The Role of Transfer RNA
1. Transfer RNA (tRNA) molecules transfer amino acids to the ribosomes.
2. The tRNA is a single-stranded ribonucleic acid that doubles back on itself to create regions where complementary bases are hydrogen-bonded to one another.
3. The amino acid binds to the 3’ end; the opposite end of the molecule contains an anticodon that binds to the mRNA codon in a complementary fashion.
4. There is at least one tRNA molecule for each of the 20 amino acids found in proteins.
5. There are fewer tRNAs than codons because some tRNAs pair with more than one codon; if an anticodon contains a U in the third position, it will pair with either an A or G—this is called the wobble hypothesis.
6. The tRNA synthetases are amino acid-activating enzymes that recognize which amino acid should join which tRNA molecule, and covalently joins them. This requires ATP.
7. An amino acid–tRNA complex forms, which then travels to a ribosome to “transfer” its amino acid during protein synthesis.
B. The Role of Ribosomal RNA
1. Ribosomal RNA (rRNA) is produced from a DNA template in the nucleolus of the nucleus.
2. The rRNA is packaged with a variety of proteins into ribosomal subunits, one larger than the other.
3. Subunits move separately through nuclear envelope pores into the cytoplasm where they combine when translation begins.
4. Ribosomes can float free in cytosol or attach to endoplasmic reticulum.
5. Prokaryotic cells contain about 10,000 ribosomes; eukaryotic cells contain many times more.
6. Ribosomes have a binding site for mRNA and binding sites for two tRNA molecules.
7. They facilitate complementary base pairing between tRNA anticodons and mRNA codons; rRNA acts as an enzyme (ribozyme) that joins amino acids together by means of a peptide bond.
8. A ribosome moves down the mRNA molecule, new tRNAs arrive, the amino acids join, and a polypeptide forms.
9. Translation terminates once the polypeptide is formed; the ribosome then
dissociates into its two subunits.
10. Polyribosomes are clusters of several ribosomes synthesizing the same protein.
11. To get from a polypeptide to a function protein requires correct bending and twisting; chaperone molecules assure that the final protein develops the correct shape.
12. Some proteins contain more than one polypeptide; they must be joined to achieve the final three-dimensional shape.
C. Translation Requires Three Steps
1. During translation, mRNA codons base-pair with tRNA anticodons carrying specific amino acids.
2. Codon order determines the order of tRNA molecules and the sequence of amino acids in polypeptides.
3. Protein synthesis involves initiation, elongation, and termination.
4. Enzymes are required for all three steps; energy (ATP) is needed for the first two steps.
5. Chain Initiation
a. A small ribosomal subunit attaches to mRNA in the vicinity of the start codon (AUG).
b. First or initiator tRNA pairs with this codon; then the large ribosomal subunit joins to the small subunit.
c. Each ribosome contains three binding sites: the P (for peptide) site, the A (for amino acid) site, and the E (for exit) site.
d. The initiator tRNA binds to the P site although it carries one amino acid, methionine.
e. The A site is for the next tRNA carrying the next amino acid.
f. The E site is to discharge tRNAs from the ribosome.
g. Initiation factor proteins are required to bring together the necessary translation components: the small ribosomal subunit, mRNA, initiator tRNA, and the large ribosomal subunit.
6. Chain Elongation
a. The tRNA with attached polypeptide is at the P site; a tRNA-amino acid complex arrives at the A site.
b. Proteins called elongation factors facilitate complementary base pairing between the tRNA anticodon and the mRNA codon.
c. The polypeptide is transferred and attached by a peptide bond to the newly arrived amino acid in the A site.
d. This reaction is catalyzed by a ribozyme, which is part of the larger subunit.
e. The tRNA molecule in the P site is now empty.
f. Translocation occurs with mRNA, along with peptide-bearing tRNA, moving to the P site and the spent tRNA moves from the P site to the E site and exits the ribosome.
g. As the ribosome moves forward three nucleotides, there is a new codon now located at the empty A site.
h. The complete cycle is rapidly repeated, about 15 times per second in Escherichia coli.
i. The ribosomes will reach a stop codon, termination will occur, and the peptide will be released.
7. Chain Termination
a. Termination of polypeptide synthesis occurs at a stop codon that does not
code for amino acid.
b. The polypeptide is enzymatically cleaved from the last tRNA by a release factor.
c. The tRNA and polypeptide leave the ribosome, which dissociates into its two subunits.
d. Proteomics is a new field of biology that aims to understand protein structures, and the functions of metabolic pathways.
D. Gene Expression
Gene expression involves transcription, translation, and protein activation.
The first few amino acids of a polypeptide act as a signal peptide that indicates where the polypeptide belongs in the cell or if it is to be secreted by the cell.
2. After the polypeptide enters the lumen of the ER, it is folded and further processed by addition of sugars, phosphates, or lipids.
3. Transport vesicles carry the proteins between organelles and to the plasma membrane.
12.6 Structure of Eukaryotic Chromosomes and Genes
The DNA is wound around a core of eight protein molecules (“beads on a string”); the proteins are called histones and each “bead” is called a nucleosome.
Histones play a structural role in chromosome structure and package the large DNA into the small nucleus.
The nucleosomes also contribute to the shortening of DNA by folding it into a “zigzag” structure.
During interphase, some chromatin is highly compact, darkly stained, and genetically inactive heterochromatin.
The rest is diffuse, lightly colored euchromatin thought to be genetically active.
Euchromatin activity is related to the extent nucleosomes are coiled and condensed.
A nucleosome is a bead-like unit made of a segment of DNA wound around a complex of histone proteins.