ESSENTIAL KNOWLEDGE 3.A.1:

DNA, and in some cases RNA, is the primary source of heritable information.

iv. tRNA brings the correct amino acid to the correct place on the mRNA.

v. The amino acid is transferred to the growing peptide chain.

vi. The process continues along the mRNA until a “stop” codon is reached.

vii. The process terminates by release of the newly synthesized peptide/protein.

d. Phenotypes are determined through protein activities. To foster student understanding of this concept, instructors

can choose an illustrative example such as: • Enzymatic reactions • Transport by proteins • Synthesis • Degradation e.

GENETIC INFORMATION IS TRANSMITTED FROM ONE GENERATION TO THE NEXT THROUGH DNA OR RNA.

Genetic information is stored in and passed to subsequent generations through DNA molecules and, in some cases, RNA molecules.

RNA WORLD HYPOTHESIS The RNA world

hypothesis proposes that self-replicating ribonucleic acid (RNA) molecules were precursors to current life.

RNA stores genetic information like DNA, and catalyzes chemical reactions like an enzyme protein.

Many viruses also store and transmit RNA

CHROMOSOMES Noneukaryotic organisms have circular

chromosomes, while eukaryotic organisms have multiple linear chromosomes, although in biology there are exceptions to this rule.

linear circular

PROKARYOTES, VIRUSES AND EUKARYOTES CAN CONTAIN PLASMIDS, WHICH ARE SMALL EXTRA-CHROMOSOMAL, DOUBLE-STRANDED CIRCULAR DNA MOLECULES.

THE PROOF THAT DNA IS THE CARRIER OF GENETIC INFORMATION INVOLVED A NUMBER OF IMPORTANT HISTORICAL EXPERIMENTS. THESE INCLUDE:i. Contributions of Watson, Crick, Wilkins, and

Franklin on the structure of DNAii. Avery-MacLeod-McCarty experimentsiii. Hershey-Chase experiment

THE GREAT DEBATEWhich chemical is used to store and transmit

genetic information?

Protein or DNA

Most Scientists of the day(early and mid 1900(s) agreed that the substance must be

protein.

EVIDENCE FOR DNA AS GENETIC MATERIAL Griffith, 1928 - In his work with

Streptococcus pneumoniae, Griffith realized that some “transforming” agent was exchanged between bacteria which enabled to acquire traits from one another.

The use of heat to inactivate cells suggested that the agent was not protein.

This phenomenon is now called transformation - a change in phenotype by taking genetic material from the environment.

GRIFFITH EXPERIMENT

AVERY-MACLEOD-MCCARTY EXPERIMENTS Took Griffiths experiment a step further by

isolating different chemical to see which one would transform bacteria.

Avery, et al., 1944 - isolated various chemicals from bacteria and used them to try transform bacteria. Only DNA worked.

VIRUSES ARE MADE OF NUCLEIC ACID AND PROTEIN

HERSHEY CHASE EXPERIMENT (1952)

CHARGAFF (1947)

Adenine pairs Thymine; Cytosine pairs Guanine

If a mixture made from cells contained 20% Adenine, then what is the percentage of Guanine?

STRUCTURE OF DNA Wilkins and

Franklin used X-ray diffraction to attempt to find the structure of DNA.

THE STRUCTURE OF DNA WAS DISCOVERED

Watson and Crick (1953)

Double Helix Sides: phosphate

and sugar Rungs:

nitrogenous bases held together by hydrogen bonds

DNA NUCLEOTIDE – MONOMER OF DNA AND RNA

OO=P-O O

Phosphate Group

NNitrogenous base (A, G, C, or T)

CH2

O

C1C4

C3 C2

5

Sugar(deoxyribose)

NITROGENOUS BASES

Double ring PURINESAdenine (A)Guanine (G)

Single ring PYRIMIDINESThymine (T)Cytosine (C)

T or C

A or G

DNA STRANDS ARE ANTI-PARALLEL

P

P

P

O

O

O

1

23

4

5

5

3

3

5

P

P

PO

O

O

1

2 3

4

5

5

3

5

3

G C

T A

STRUCTURE OF DNA

DNA REPLICATION ENSURES CONTINUITY OF HEREDITARY INFORMATION. Replication is a semiconservative process;

that is, one strand serves as the template for a new, complementary strand.

DNA REPLICATION (SEMICONSERVATIVE MODEL)

ORIGIN OF REPLICATION Origin of replication (“bubbles”): beginning of

replication Replication fork: ‘Y’-shaped region where new strands

of DNA are elongating Helicase:catalyzes the untwisting of the DNA at the

replication fork DNA polymerase:catalyzes the elongation of new DNA

DNA REPLICATION

Antiparallel nature: • sugar/phosphate backbone runs in opposite directions (Crick); • one strand runs 5’ to 3’, while the other runs 3’ to 5’; • DNA polymerase only adds nucleotides at the free 3’ end, forming new DNA strands in the 5’ to 3’ direction only

DNA REPLICATION Leading strand:

synthesis toward the replication fork (only in a 5’ to 3’ direction from the 3’ to 5’ master strand)

Lagging strand: synthesis away from the replication fork (Okazaki fragments); joined by DNA ligase (must wait for 3’ end to open; again in a 5’ to 3’ direction)

Initiation: Primer (short RNA sequence~w/primase enzyme), begins the replication process

SIMILAR TO ATP!! The energy to add new nucleotides comes from the substrates themselves which are nucleoside triphosphates.

The loss of two phosphates from the substrate provides the energy to drive the reaction.

RETROVIRUSES REVERSE THE NORMAL FLOW OF GENETIC INFORMATION Genetic information in retroviruses is a

special case and has an alternate flow of information: from RNA to DNA, made possible by reverse transcriptase, an enzyme that copies the viral RNA genome into DNA.

This DNA integrates into the host genome and becomes transcribed and translated for the assembly of new viral progeny.

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RNA

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RNA DIFFERS FROM DNA1. RNA has a sugar ribose

DNA has a sugar deoxyribose2. RNA contains the base uracil (U)

DNA has thymine (T)3. RNA molecule is single-stranded

DNA is double-stranded

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STRUCTURE OF RNA

31

. THREE TYPES OF RNA

Messenger RNA (mRNA) carries genetic information to the ribosomes

Ribosomal RNA (rRNA), along with protein, makes up the ribosomes

Transfer RNA (tRNA) transfers amino acids to the ribosomes where proteins are synthesized

THE 4TH TYPE OF RNA! THE ROLE OF RNAI INCLUDES REGULATION OF GENE EXPRESSION AT THE LEVEL OF MRNA TRANSCRIPTION.

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gen expression, typically by causing the destruction of specific mRNA molecules.

Protein Synthesis

CENTRAL DOGMA Genetic information flows from a sequence of

nucleotides in a gene to a sequence of amino acids in a protein.

THE TRIPLET CODE

The genetic instructions for a polypeptide chain are ‘written’ in the DNA as a series of 3-nucleotide ‘words’

Codons ‘U’ (uracil) replaces

‘T’ in RNA

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NAME THE AMINO ACIDS

GGG?UCA?CAU?GCA?AAA?

TRANSCRIPTION The enzyme RNA-polymerase reads the DNA

molecule in the 3' to 5' direction and synthesizes complementary mRNA molecules that determine the order of amino acids in the polypeptide.

TRANSCRIPTION RNA polymerase: pries

DNA apart and hooks RNA nucleotides together from the DNA code

Promoter region on DNA: where RNA polymerase attaches and where initiation of RNA begins

Terminator region: sequence that signals the end of transcription

Transcription unit: stretch of DNA transcribed into an RNA molecule

TRANSCRIPTION Initiation~ transcription

factors mediate the binding of RNA polymerase to an initiation sequence (TATA box)

Elongation~ RNA polymerase continues unwinding DNA and adding nucleotides to the 3’ end

Termination~ RNA polymerase reaches terminator sequence

MRNA MODIFICATIONS In eukaryotic cells the mRNA transcript

undergoes a series of enzyme-regulated modifications.

• Addition of a poly-A tail• Addition of a GTP cap• Excision of introns

MRNA MODIFICATION 1) 5’ cap: modified guanine; protection; recognition site for

ribosomes 2) 3’ tail: poly(A) tail (adenine); protection; recognition;

transport 3) RNA splicing: exons (expressed sequences) kept,introns

(intervening sequences) spliced out; spliceosome

TRANSLATION Translation of the mRNA occurs in the

cytoplasm on the ribosome. In prokaryotic organisms, transcription is

coupled to translation of the message. Translation involves energy and many steps, including initiation, elongation and termination.

TRANSLATION mRNA from

nucleus is ‘read’ along its codons by tRNA’s anticodons at the ribosome

tRNA anticodon (nucleotide triplet); amino acid

TRANSLATION rRNA

site of mRNA codon & tRNA anticodon coupling

P site holds the tRNA carrying the growing polypeptide chain

A site holds the tRNA carrying the next amino acid to be added to the chain

E site discharged tRNA’s

TRANSLATION Initiation~

union of mRNA, tRNA, small ribosomal subunit; followed by large subunit

Elongation~ •codon recognition •peptide bond formation •translocation

Termination~ ‘stop’ codon reaches ‘A’ site

Polyribosomes: translation of mRNA by many ribosomes (many copies of a polypeptide very quickly)

MUTATIONS: GENETIC CHANGES IN A CELL

Point mutations…. Changes in 1 or a few base

pairs in a single gene Base-pair substitutions: •silent

mutations no effect on protein

•missense ∆ to a different amino acid (different protein)

•nonsense ∆ to a stop codon and a

nonfunctional protein Base-pair insertions or

deletions: additions or losses of nucleotide pairs in a gene; alters the ‘reading frame’ of triplets~frameshift mutation

Mutagens: physical and chemical agents that change DNA

GENETIC ENGINEERING TECHNIQUES CAN MANIPULATE THE HERITABLE INFORMATION OF DNA AND, IN SPECIAL CASES, RNA. • Electrophoresis • Plasmid-based transformation • Restriction enzyme analysis of DNA • Polymerase Chain Reaction (PCR)

RESTRICTION ENZYMES ANALYSIS A restriction enzyme (or restriction

endonuclease) is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites.

Fragments are separated using gel electrophoresis

ELECTROPHORESIS The separation of DNA fragments according

to charge and size. Smaller fragments migrate through the jell at

a faster rate than larger fragments. The gels are stained an viewed under UV

light.

PCR The polymerase chain reaction (PCR) is a

biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications

PLASMID BASED TRANFORMATION

GENETICALLY ENGINEERED PRODUCTS: Genetically modified foods Transgenic animals Cloned animals Pharmaceuticals, such as human insulin or

factor X

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