Unit 1 DNA and Gene Expression

5 criteria of life

- Respond to environment

- cellular structure

- Replication

- Energy

- Homeostasis

Dr. Roberts 5 rules

#1: "Nothing in biology makes sense, save in

the light of evolution"

#2: Living organisms maintain

(within limits) a balanced internal environment

#3: If you move solute(s), water will follow

#4: Shape determines how the

molecule functions in its environment

#5: Organisms have limited access to building blocks and are energy-limited

Explain Mendel's laws of inheritance

- law of segregation: alleles of a gene separate equally during meiosis

- law of independent assortment: Each chromosome sorts independently during Meiosis I

- genes are associated with specific traits

How is genetic information stored in chromosomes?

Nucleosome --> chromatin --> scaffold chromatin --> condensed heterochromatin --> condensed chromosome

what are the experiment's that proved DNA as the molecule of hereditary

1. Griffith - dead bacterial cells can genetically transform living bacteria cells

2. Avery, McLeod, McCarthy - Transforming substance is DNA

3. Hershey & Chase - demonstrated DNA was primary genetic material

General structure of nucleotide

Phosphate group + pentose sugar (ribose or deoxyribose) + nitrogenous base

5' = where phosphate is attached

3' = -OH group

What is Chargraff's rule? and how did they effect the secondary structure of DNA?

Purines are adenine and Guanine and pyrimidines are cytosine and thymine

- Base pairs A & T go together

- Base pairs C & G go together

therefore the amount of A in DNA is equal to the amount of T and same thing for C & G

- There is a difference in number of hydrogen bonds btw. base pairs giving rise to major and minor grooves

- In the major groove there is lots of space so that is where most protein interactions occur

Events of DNA replication

- helicase unzips the DNA

- ssBP bind to DNA to stabalize the strands and keep them from connecting back together

- primase add short RNA primer so DNA polymerase can start adding base pairs (5' --> 3')

- Rna primers are removed and replaced with DNA

- Dna ligase glues okazaki fragments

Proofing:

- Dna poly. checks for mistakes as it works

leading vs lagging strand

Problem: DNA polymerase can only add nucleotides in a 5' to 3' direction

Leading strand:

- template strand that runs from 3' to 5' so Rna primer is only added once and dna poly. moves towards the replication fork continously

Lagging strand:

- template strand runs from 5'-->3' and dna poly cannot synthesize in this direction

- does not synthesize toward fork and instead works in short bursts creating okazaki fragments

- Each fragment requires a new RNA primer

Semiconservative

method of replication that implies that each new strand of DNA is half original and half new. This concept was found through the messelson-stahl experiment with heavy and light nitrogen. samples were a mix of heavy and light due to perserving the original parent strand of the heavy nitrogen.

Prokaryotic vs. Eukaryotic replication:

SIMILARITIES

- both bi-directional processes

- both require primers to start the process

- both have leading and lagging

- DNA polymerase enzymes work from the direction of '5-'3 so new nucleotides are added to the '3 end of a primer.

DIFFERENCES

- prokaryotes only have one origin of replication

- prokaryotes do not have the telomere shortening problem since their DNA is circular

Function of telomerase:

- eukaryotes have linear chromosomes and dna poly. cannot add nucleotides to the very end of the lagging strand, leaving a short section un-copied

- telomerase allows to extend dna by binding to the 3' overhang

- synthesizing in 5' --> 3', multiple telomere repeats are added, extending the chromosome end

- once the end is extended, primase adds rna primer and dna polymerase fills in the complementary strand

- active in many cancer cells, stem cells, germ cells

Topoisomerase:

Enzyme that functions in DNA replication, helping to relieve strain in the double helix ahead of the replication fork. DNA replication cannot occur without this enzyme

DNA polymerase:

- requires a free OH group

- adds to the growing strand as triphosphates, which release pyrophosphates, providing the energy to power the reaction

Explain transcription

- one strand of DNA is used as template

1. initiation:

- gene begins at promoter, which tells RNA polymerase where to start, which strand to copy, and which direction to move

Eukaryotes: promotor includes TATA box, transcription factors bind first, and then RNA polymerase

- RNA polymerase locally unwinds DNA creating a transcription bubble

Synthesis:

- RNA polymerase synthesizes from 5' --> 3'

- Uracil replaces thymine

- RNA strand peels away from template strand as it grows

- NO PRIMER NEEDED

Termination:

- stops when RNA poly. reaches termination sequence and detaches from dna

-In prokaryotes:

Termination signals are part of the RNA itself

In eukaryotes:

Transcription continues past the gene

RNA is later cut during processing

RNA Processing (EUKARYOTES ONLY)

- before processed it is called pre-mrna

1. 5' cap: modified guanine nucleotide is added to the 5' end to protect, helps with recognition, and assists with export from nucleus

2. Poly-A-Tail: long chain of adenine added to 3' end to increase stability, nuclear export, and affects how long the mrna lasts

3. Splicing: Econs remain (exit nucleus), intron are spliced out (stay in nucleus)

- introns removed by spliceosomes

- allows for one gene to form multiple proteins if exons are rearranged or alternatively spliced

Result: mature mRNA leaves nucleus and goes to ribosome

List and define the key molecules in transcription and translation

Transcription:

- RNA Polymerase

- Transcription factors (eukaryotes) - help RNA poly. bind to promotor

- Pre-mRNA and mRNA

- Spliceosomes: remove introns

Translation:

- Ribosomes

- rRNA, mRNA, tRNA (transfer rna)

- Amino acids: building blocks of proteins.

- Codon: A three-nucleotide sequence on mRNA which specifies one amino acid or a stop signal

- Anticodon: A complementary three-nucleotide sequence on tRNA. Ensures the correct amino acid is added.

- Start codon: AUG (methionine)

- Stop codon: UAG, UGA, UAA (DO NOT CODE FOR AMINO ACID)

What is the central dogma?

DNA --> RNA --> Protein

gene expression in prokaryotes vs. eukaryotes

Differ in 7 major ways:

1. The RNA polymerases are different

2. Prokaryotic cells only have one RNA polymerase, eukaryotic cells have three.

3. Eukaryotes depend on transcription factors.

4. Transcription is terminated differently; in prokaryotic cells the RNA Pol just falls off when ready to terminate, but in eukaryotic cells, the RNA must be cleaved off while RNA Pol continues to transcribe.

5. Eukaryotic RNA must be processed to be functional.

6. Prokaryotic ribosomes are smaller than Eukaryotic ribosomes.

7. In prokaryotic cells transcription and translation can happen simultaneously. Eukaryotic compartmental organization separates transcription from translation.

Deletions/insertions

cause more dramatic changes in the DNA, including frameshift mutations, and often result in complete loss of gene function.

duplications, translocations, and inversions

Duplications: Segment of chromosome is copied and repeated, resulting in extra genetic material (happens between homologous chromosomes)

Translocations: A piece of one chromosome breaks off and attaches to a different chromosome (happens btw. non-homologous pairs)

Inversions: Chromosome segment breaks, flips, and reinserts itself in reverse orientation

Nonsense vs Missense mutation

Missense: changes one base, so that a different amino acid is added to the protein

- Conservative: proteins function is not changed

- non-conservative: proteins function does change

Nonsense: changes a codon that codes for an amino acid into a stop codon

- translation stops early and produces a shortened protein, typically nonfunctional

How can cytosine mutate into thymine

A chemical change called deamination (removal of amino group). when cytosine amino group is removed it turns into uracil. if replication occurs before repair uracil pairs with adenine and then during the next round of replication A bind with T

- methylated cytosine

How do gene mutations cause PKU?

- caused by mutation in PAH, converting phenylalanine into tyrosine; phenylalanine build up is toxic

- single trait autosomal recessive

How do gene mutations cause sickle cell disease?

- JUST one nucleotide swap (A --> U ; glutamine to valine) in hemoglobin protein and RBCs become sickle shaped

- single trait autosomal recessive w/ incomplete dominance

- if you are heterozygote you have sickle cell trait which gives you defense against malaria

Explain how aneuploidy occurs through nondisjunction

- nondisjunction is when chromosomes fail to seperate properly, and both copies go to same daughter cell

Meiosis I: n+1, n+1, n-1, n-1

Meiosis II: n,n, n+1, n-1

Differences of Sexual Development (DSD)

Turner syndrome: individual has only one X chromosome from nondisjunction during meiosis (females)

Klinefelter syndrome: XXY (males) caused by nondisjunction

Guevedoce: Mutation of the 5-alpha reductase enzyme

Structure of tRNA

- single stranded RNA that folds back on itself from intramolecular base pairing

- Acceptor stem: 3' end (CCA), amino acid attaches here

- contains 3 nucleotide anticodon

tRNA is a single-stranded RNA that folds into a cloverleaf secondary structure and an L-shaped tertiary structure, with an anticodon for mRNA recognition and a 3′ acceptor stem that binds a specific amino acid.

EPA site on ribosome

A: arrive - hold the incoming tRNA carrying the next amino acid

P: process - holds the tRNA carrying the growing polypeptide chain

E: Exit - holds the tRNA after it has released its amino acid

Explain the process of translation?

initiation: Small ribosomal subunit binds to the mRNA near the start codon.

The start codon AUG is recognized.

An initiator tRNA carrying methionine binds to AUG.

This tRNA binds directly to the P site.

The large ribosomal subunit joins, forming a complete ribosome.

Elongation:

A charged tRNA with the correct anticodon enters the A site.

Base pairing ensures the correct amino acid is selected.

* codon and anticodon interactions only occur at A & P site

Termination:

A stop codon (UAA, UAG, or UGA) enters the A site.

No tRNA matches a stop codon.

A release factor binds instead.

The completed polypeptide is released.

Ribosomal subunits separate from the mRNA.

- poly peptide may undergo post-translational processing (adding phosphates or sugars, etc.)

Retroviruses?

RNA --> DNA and insert that DNA into hosts genome and directs the production of new viruses.

- carry enzyme reverse transcriptase

Still learning (2)

You've started learning these terms. Keep it up!

Law of segregation:

alleles of a gene separate equally during meiosis

Law of independent assortmnet

each chromosome sorts independently during meiosis I