Bio Unit 3- Molecular Genetics (Chapter 5)

Nucleotide

One individual unit/building block of DNA or RNA, made up of

• one nitrogenous base of any kind

• a sugar

• phosphate group (on the sugar)

Erwin Chargaff’s experiment (Chargaff’s Rule)

Chargaff discovered the Chargaff’s Rule (base pairing) by looking at the chemical make up of DNA. He found that:

• Adenine is always found with Thymine (and vice versa)

• Guanine is always found with Cytosine (and vice versa)

Complementary Base Pairing

The pairing of complementary bases between the two strands of DNA as shown by Chargaff’s rule

Anti-parallel

Refers to the orientation of the two DNA strands against each other. They are parallel to each other but face opposite directions, meaning that when one strand runs 5' to 3', the other strand runs 3' to 5'. Referring to the carbons on the sugar from the ether group, from top to bottom of the strand.

genome

the total genetic material of an organism, the “Instruction book” on the entire body in DNA

gene

A distinct functional region within DNA that holds genomes.

Frederick Griffith’s Experiment

tested 2 forms of the same bacterium on mice: an S-strain that was pathogenic (carries the virus that would kill the mice) but could be made non-pathogenic by heating it, the other, R-strain, being non-pathogenic and harmless. When he injected the heated S strain and the R strain into the mice, the mice died. This indicated that there was something being transferred from the dead bacteria in the S-strain to the R-strain. This transfer is referred to as transformation.

Transformation

The movement of genetic material from a dead organism to a live one, altering their DNA. Found in Griffith’s Experiment

Initiation Phase

First phase of semi-conservative replication. A portion of the double helix is unwound to expose the bases for new base pairing

Elongation Phase

Phase 2 of semi-conservative replication. 2 new strands of DNA are put together using the individual parent strands as a template. The new DNA molecules (made of one parent and one new strand) wind back into helix shape.

Termination Phase

Last phase of semi-conservative replication. The new DNA molecules separate from each other completely. The replication machine is dismantled

Origin of Replication

The specific nucleotide sequence at which the process of replication starts.

Helicase

Type of enzymes that unwind and separate the double-stranded DNA during semi-conservative replication.

They cleave the h-bonds between the complementary base pairs to allow the strands to separate.

Semi conservative replication

The proved method of DNA replication in which each new DNA molecule consists of one original strand and one newly synthesized strand due to each new strand being made by using a parent strand as a guideline

DNA polymerase III

The enzyme that catalyzes the addition of new nucleotides one at a time to create a new strand based off of the parent strand.

• only attaches to the 3’ hydroxyl end of pre-existing nucleotides

• Can only synthesize a new strand from a parent strand in the 5’ to 3’ direction

primer

The short strands of RNA used to start the replication process by providing a free 3' hydroxyl group for the nucleotide addition done by DNA polymerase III

Okazaki fragments

Short DNA fragments created when a nucleotide is added to the various primers of the lagging strand

The use of primers means that the fragments always start with an RNA section

lagging strand

The second strand, ordered from 5’ to 3’ (forces the new strand to be made from 3’ to 5’)

needs several primers for replication which leads to the creation of Okazaki fragments

leading strand

needs only one primer for DNA replication

ordered 3’ to 5’ (allows the new strand to be from 5’ to 3’)

DNA polymerase I

• enzyme that removes the RNA primers from the Okazaki fragments of the lagging strand, and fills that space by extending the neighboring DNA nucleotide. This causes the Okazaki fragments to be just made of DNA which are then joined together by DNA ligase

• can also recognize mistakes in replication, cutting out the incorrect or extra base from the new strand and replacing it with the correct one (works with DNA polymerase II to do this) using the parent strand as a template

DNA ligase

the enzyme that catalyzes the joining together of Okazaki fragments that have only DNA nucleotides

Creates a continuous DNA strand

replication machine

protein-DNA complex at the end of each replication fork that carries out replication

DNA polymerase II

recognizes mistakes in replication and cuts out the incorrect base from the new strand, replacing it with the correct one using the parent strand as a template

Mismatch repair

A replication error fixing method in which specialized enzymes recognize the deformities in newly formed molecules that have mismatched base. These enzymes then bind to the DNA to remove the incorrect base from the new strand and replace it with the correct one

Errors in replication that pass the DNA polymerase and mismatch repair are called what?

Mutations

telomere

A repetitive DNA sequence at the end of a strand added to avoid the loss of important genetic information when the RNA section of the very last Okazaki fragment is removed and cannot be replaced

one gene/one polypeptide hypothesis

The theory that each gene only holds the information needed to produce a single polypeptide.

mRNA

• “Messenger RNA”

• RNA that contains the genetic information of a gene and carries it to the protein synthesis machinery

  • provides information that determines the amino acid sequence of a protein

  • acts as an intermediary

genetic code

• a set of rules for determining how genetic information in the form of nucleotides are transferred into amino acid sequences in proteins.

• a code specifying the relationship between a nucleotide codon and an amino acid

Triplet Hypothesis

• Only 4 nucleotides in RNA, but 20 different amino acids. To produce 20 different combination, a minimum of three nucleotides needs to be used (4 times 4 times 4 = 64 vs 4 times 4 = 16) leading to the name

• States that genetic code consists of 3 different nucleotides

gene expression

• the transfer of genetic information from DNA to RNA to protein, leading to it determining the phenotype (and being expressed)

transcription

• the transferring of genetic information from DNA template to RNA

  • the mRNA is synthesized using the DNA as a template

translation

• the transferring of genetic information from mRNA to protein

  • the protein is synthesized using mRNA as a template

Alfred Hershey and Marsha Chase experiment

• proved that DNA is the hereditary material not protein by using a virus called T2 bacteriophage

  • They tracked the DNA and protein molecules by using radioisotopes (³⁵S for proteins and ³²P for DNA).

  • When the ³⁵S containing virus infected the bacteria, they found after agitation and separation that the radioactive material was only in the liquid medium, and not in the bacteria

  • When the ³²P containing virus infected the bacteria, they found that the radioactive material was inside the bacteria, confirming that DNA is the genetic material as it was the only material to be able to enter the bacteria in order to alter it

What is T2 bacteriophage made out of?

protein coating over nucleic material

Rosalind Franklin’s experiment

• used X-ray diffraction to study the structure of DNA

• took X-Ray images of DNA molecules, and used their background with mathematics to calculate the diffractions in the image, and figured out what it would look like without the diffraction: arriving at the shape of the DNA that is known today

• However they were not credited for this discovery as Watson and Crick stole their work to use for their own model and had it published before them

James Watson and Francis Crick

• Put together the information and discoveries of all of the experiments (stolen from franklin) and created the first 3D DNA model that could be seen and understood by the general public

• Proposed the double helix formation that allows for difference in DNA shape among species

Oswald Avery’s experiment

• Used the process of elimination to provide further proof of Frederick Griffith’s conclusion

• He subjected three separate identical heat killed S-strain bacteria to different enzymes (one for protein, one for DNA and one for RNA)

• The enzyme treated extracts were then mixed with non-pathogenic R-strains

• If the R-strains were transformed into pathogenic S-strains, then this meant that the hereditary material had not been destroyed by the enzyme, and that the target was wrong

• The only R-strain that did not transform into a pathogenic S-strain was the one afflicted by the DNA enzyme, proving that the hereditary material that determines whether the strain is pathogenic is made of DNA

  • Therefore all genetic material is made of DNA

Spontaneous mutation

type of mutation that takes place when something in the replication process goes wrong (ex: strand slippage)

Induced mutation

• a mutation that takes place due to the environment (ex: cigarettes, radiation)

  • Two types:

    • chemical mutagen

    • physical mutagen

Point Mutation

  type of single gene mutation that involves the SUBSTITUTION of one nucleotide for another.

• If a substitution changes the amino acid, it’s called a MISSENSE mutation 

• If a substitution does not change the amino acid, it’s called a SILENT mutation

• If a substitution changes the amino acid to a “stop,” it’s called a NONSENSE mutation

Missense Mutation

A point mutation that changes the amino acid

Silent Mutation

  A point mutation that does not change the amino acid

mutagen

• substance that causes mutations

• Two types: chemical and physical

mutation

Nonsense mutation

• A point mutation that changes the amino acid to a “stop”

Frameshift

• a type of single gene mutation that involves INSERTION or DELETION of a single base pair.

Single gene mutation

mutation that involves the change in nucleotide sequence of one gene

Chemical mutagen

• Type of induced mutation

• Molecules

• enters the nucleus and reacts with DNA to induce a mutation

Physical mutagen

• Type of induced mutation

• an event or a substance (ex: high energy radiation like UV rays or X-rays )that changes the physical structure of DNA, causing a mutation

Photorepair

• specific type of DNA repair specialized for fixing damage caused by UV radiation (binds thymines together covalently into a dimer, altering the shape of DNA)

• photolyase enzyme recognizes the damage, binds to the dimer, and uses visible light to cleave the thymine dimer such that the covalent bond is broken

Excision Repair

• a non-specific type of DNA repair that can fix a variety of damage through excision (cutting out) of the damaged or incorrect base to later be replaced by DNA polymerase

Specific Repair Mechanism

• a DNA repair mechanism that is specialized to repair one specific type of damage

• Ex: photorepair

Non-specific Repair Mechanism

• A DNA repair mechanism that is versatile and capable of fixing multiple kinds of damage done to the DNA molecule

• Ex: Excision Repair

Gene Regulation

• the control of how much each gene is expressed

• determines which genes are active or inactive and how active they are (how much protein is produced)

Constitutive Genes

• Also called “housekeeping” genes

• Essential for survival and therefore always active unlike most genes

What are the three different levels of DNA replication of prokaryotes that allows for gene regulation?

• Transcription

• Translation

• After a protein has been synthesized

Operon

• Found in prokaryotes

• a region in which a cluster of genes are under the control of one promoter

• Contains:

  • a promoter

  • an operator

  • structural genes

lac operon

• where the genes that encode the enzymes necessary to break down the sugar lactose are located

• Found in E.coli chromosome

• inducible operon: activated when lactose is present

The Coding Region

• the portion of DNA that contains structural genes required for protein synthesis

  • in lac operon, the structural genes for lactose are found here

Regulatory Region

• the portion of DNA that contains genes and compartments that allow gene regulation to take place

  • the specific compartments and genes depend on the chromosome

What does the coding region of a lac operon contain?

• structural genes that allow the synthesis for 3 different enzymes that break down lactose

What does the regulatory region of a lac operon contain?

• A promoter

• An operator

• A CAP binding site

Promoter

• a sequence of special nucleotides that indicate where RNA polymerase should bind to initiate transcription

Operator

• a DNA sequence that a repressor protein binds to to inhibit transcription initiation

CAP site

• a DNA sequence that a CAP (catabolize activation protein) binds to to increase the rate of transcription not necessary but useful when a lot is needed fast)

How and when is the lac operon deactivated?

• when there is no lactose, and therefore the enzymes needed to break it down are not required

• Deactivated by repressor that binds to the operator, preventing RNA synthase from doing so and initiating transcription

How and when is the lac operon activated?

• when there is lactose which needs to be broken down, and allolactose is made

• Activated through the allolactose binding to the repressor, preventing the repressor from binding to the operator

  • the operator becomes available for RNA synthase to bind to it

• The binding of a CAP protein can further increase the rate at which transcription takes place

Inducible operon

• an operon that can be activated when a product is present

trp (tryptophan) operon

• always active unless a repressor is present

• synthesizes enzymes that are required to produce tryptophan

What is a trp operon made of structurally?

• a coding region

  • contains 5 different genes that produce enzymes that make tryptophan

• a regulatory region that contains

  • a promoter

  • an operator

When and how is a trp operon activated?

• under normal conditions a trp operon is always active because tryptophan is always necessary and must be synthesized

• since the synthesized tryptophan is being used, it does not bind a repressor protein and the synthesis continues

When and how is a trp operon deactivated?

• when there is a high concentration of tryptophan

• Because there is a lot of tryptophan, it is more likely that the tryptophan binds to a repressor protein. Once it does, this repressor binds to the operator of trp operon to prevent RNA synthase from moving to the genes from the promoter by binding to it, thus deactivating the operon

Structural Genes

genes in an operon that code for the product protein

Restriction enzyme/endonuclease

• An enzyme found in bacteria that recognizes a target sequence in the DNA of a virus that tries to inject its DNA inside the bacteria to take it over, and cuts it off

• Used by scientists to cut up DNA such that only the desirable portions remain and can be added to other strands of DNA to create transgenic organisms

Restriction Site

• The specific point of the DNA at which the restriction endonuclease cuts staggerdly , separating all except the base pairs at each end

What are the characteristics of restriction endonuclease that make it useful for scientists

• Specificity

  • can target specific portions of the DNA to cut, which usually has the same base pattern but inverted between the strands

• Staggered Cuts

  • cuts in a staggered pattern that leaves behind sticky ends which are unpaired bases that are useful in bonding the fragment with a fragment of DNA from a different organism

Sticky ends

• unpaired bases at the end of DNA fragments caused by the staggered cut of restriction endonuclease

How is a recombinant DNA molecule made?

• Two DNA molecules from different organisms are cut with the same restriction endonuclease

  • the endonuclease being the same causes them to have the same sticky ends, complimenting each other

  • the endonuclease is chosen specifically so that it can cut both DNA molecules

• Base pairing between the sticky ends brings the two DNA molecules together

• DNA ligase covalently joins the two strands

Repressible operon

• an operon that is always active, and can only be deactivated by a high amount of the protein it produces

Enhancer

• a DNA sequence that promotes transcription

• made up of distal control elements

What are the binding sites on the large sub-unit of a ribosome called in order from left to right?

Site E, Site P, Site A

In what direction do ribosomes read?

5’ to 3’

What is the difference between a regulatory sequence and a regulatory gene?

• A regulatory sequence is a sequence on the DNA molecule that a regulatory protein can bind to in order to control how much the genes that follow are expressed. ex:

  • promoter

• A regulatory gene is a gene on the DNA molecule that codes for a regulatory protein (can also be enzyme)