Cell Structure Review and Introduction to DNA

Did you know? 100 years ago we did not know why some

children had brown eyes and some blue 75 years ago we did know the structure of

dextrose 50 years ago we did not know the correct number

of chromosomes 25 years ago we did not know any of the genes

linked to cancer What is one you know of?

BRCA breast cancer gene

Differences Between Eukaryote and Prokaryote Cell

Eukaryotes Nucleus Membrane-bound

organelles Microtubules are the

building blocks for a flagella

Cell membranes can contain cholesterol

Cell size is usually bigger Introns in the DNA

Prokaryotes No nucleus No membrane-bound

organelles Flagella is the building

block No cholesterol in cell

membranes Cell size is usually smaller Usually no introns

Eukaryotes

Sources of DNA How are Eukaryotes and Prokaryotes different?

Eukaryotes DNA is in the nucleus

Prokaryotes have no nucleus, so where is the DNA? Floating in the cytoplasm, which is usually attached to the cell

membrane. Bacteria contain 1 long circular DNA molecule, super coiled. E. coli contains 1 chromosome w/4000 genes and 4.6 million base pairs

(bp) R Plasmids: bacteria with small ring of DNA floating in cytoplasm and

these contain the antibiotic resistance genes Genes are turned “on” or “off” easily

Eukaryotic DNA DNA packaged into chromosomes Each single DNA may contain several million

nucleotides and many thousands of genes Humans have 46 chromosomes per cell with

about 3 billion base pairs making up about 40,000 genes

Historical Figures in Molecular Biology (visit DNAi.org)

Miescher Griffith Avery, McCarty and MacLeod Chargaff Wilkins, Franklin, Watson & Crick

Key people in genetics and DNA Gregor Mendel: heredity passed down from parents;

relationship between phenotype and genotype Schleiden and Schwann’s “cell theory” explained fertilization

of sperm and egg to make zygote 1905 discovered sex chromosomes existed; years later realized

there are more chromosomes that are responsible for traits Watson and Crick: 3D structure of DNA in 1953

DNA actually discovered in 1869, by 1900 understood that it was composed of 5C sugar, phosphate, and 5 types of nitrogen rich bases (ATCGU), 1920s we understood that RNA and DNA were different

Key Historical People Erwin Chargaff

determined percents of purines and pyrimidines present

Rosalind Franklin X-ray diffraction technique was key to

understanding the helix structure

C

C

C

C

N

N

OO

N

CC

CC

N

N

N

N

N

C

Role of X-Ray Crystallography X-rays diffracted by the regularly arranged

atoms of a simple crystal (Max von Laue)

Pauling, Franklin, Wilkins, Watson and Crick were all working diligently to discover the structure of DNA

What Nucleotides are involved? What are the 4 nucleotides of DNA?

Adenine, thymine, guanine, cytosine

What pairs with what? Adenine and Thymine Cytosine and Guanine

The three parts of the nucleotide building block of DNA are the sugar, the base and the phosphate. The complex of the sugar with the base is called a nucleoside.

                                      

Sugar The sugar is the 5-carbon sugar deoxyribose. By convention the carbons on this sugar are labeled 1' through 5'.

Phosphate The phosphate is attached to the 5' carbon of the deoxyribose sugar.

Base The base is attached to the 1' carbon of the deoxyribose sugar. There are four different bases found in DNA. Because each base contains at least two nitrogen atoms, they are called nitrogenous bases. There are two classes of bases, the pyrimidines (cytosine (C) and thymine (T)), and the purines (adenine (A) and guanine (G)).

Complementary Base PairingDNA consists of two polynucleotide chains wound around each other to form a double helix. The two chains are held together by complementary base pairing; that is, specific bonding between A and T bases and between G and C bases on the two strands

Two antiparallel DNA polynucleotide chains held together by complementary base pairing.

To make a stable double helix, the two strands of DNA are antiparallel; that is, the 5’ - 3' direction of one strand runs opposite to the other strand.

The two DNA chains are held together by complementary base pairing between A and T bases and between G and C bases.

The helix has a right hand twist.

1.

2.

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9.

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11.

In a DNA polynucleotide chain, nucleotides are joined by phosphodiester bonds formed between the 5' carbon of one sugar and the 3' carbon of the next sugar. A free phosphate defines the 5' end of the chain and a free hydroxyl group defines the 3' end of the chain.

1. 3’ end2. phosphate3. 5’ end4. thymine5. 3’ end6. phosphodiester bond

7. cytosine8. deoxyribose9. guanine10. 5’ end

11. adenine

Basics to DNA Structure Rails of ladder: run in opposite

directions (anti-parallel) Contains alternating units of

deoxyribose sugar and phosphate Each rung composed of a base pair

held together by weak hydrogen bonds 10 base pairs per turn 34 A total so 3.4 A between pairs

DNA Replication DNA helicase Single-strand binding proteins Primase DNA polymerase DNA ligase Okazaki fragments

So why is DNA replication so important to us? DNA is the carrier of genetic information for

all living organisms Through the process of replication, the entire

genome is copied and passed down to each new cell made in the body.

Replication is also the way genetic information is passed from parents to offspring.

Replication DNA polymerase can only directly synthesize

new DNA in the 5' to 3' direction Chargaff’s Rule for determining how many

nucleotides are present: In double-stranded DNA, G = C, and A = T. If C = 21, then G = 21 and G + C is 42. Therefore A + T = 100 - 42 =

58, and T = 58/2 = 29 percent. A/T, G/C, and (A+G)/(C+T) are all equal to 1

Semiconservative Replication: making 2 daughter stands from a single parent strand Therefore DNA replication takes place prior to cell

division

DNA Helicase and SSB DNA Helicase is an enzyme which begins the unzipping process. Also prevents DNA from rebinding. Problem is that it creates a knotted up mess of DNA Topoisomerase cuts one strand of unwound and allows

it to unwind and then reseals it. It prevents damage to the DNA by allowing it to swivel.

Once DNA is unzipped the base pairs of each single stand will begin forming helix structure SSB (single strand binding proteins) are formed to

block this action; prevent recombining

Getting Replication Started

Replication Bubble

The DNA begins to split from many points along the strands and separate from that point, creating a bubble-like area.

Replication Fork

When referring to the replication of DNA in a singular direction, the original DNA splits in two, forming two prongs, which resemble a fork.

Primase and DNA Polymerase Primase is an RNA polymerase which does not

need a primer to initiate synthesis RNase H comes into remove the RNA primer made

by primase before DNA is replicated DNA polymerase III can only add nucleotides

onto the 3’ end of an existing DNA fragment so if this is the case then where does the first piece of DNA come from?

Okazaki fragments and DNA Ligase DNA synthesis is always 5’ to 3’ Leading strand is synthesized and the lagging strand has

small fragments formed which are later joined together. Fragments are called Okazaki fragments after the

scientist who discovered this process. Polymerase I removes RNA primer and replaces it with

DNA nucleotides in Okazaki fragments DNA ligase is the enzyme which joins the

Okazaki fragments together

DNA Replication

Checking your knowledge What are the two strands of DNA called after they

unzip? Leading and lagging

What enzyme is used for unzipping? DNA helicase

What direction does DNA replicate? Actually both, 5’ to 3’ easy, continuous and self

correcting; 3’to 5’ takes longer, more chance of error and requires DNA polymerase and DNA ligase

Checking your knowledge What are the fragments formed during

replication called and what strand are they formed on? Okazawki fragments; lagging 3’ to 5’ strand

What 3 enzymes are required for DNA replication? DNA helicase, DNA polymerase, topoisomerase

RNA: how is it different from DNA? Pentose sugar is ribose instead of deoxyribose Uracil replaces Thymine RNA is single stranded

What is transcription? Where does it occur? Transcription: the process of deciphering a

DNA nucleotide code and converting into into an RNA nucleotide code; RNA carries genetic message to a ribosome for translation into a protein.

Proteins do the work of cells and give cells and organisms their unique characteristics.

Note: single strand of RNA

Transcription occurs in 5’ to 3’ direction

Transcription Initiation

Transcription factors bind to the TATA box which guide the RNA Polymerase to the starting point of the gene.

Transcription Elongation

RNA Polymerase continues to assemble RNA nucleotides in a complementary fashion to the 3’5’ template strand

Transcription Termination

Once the RNA Polymerase hits the termination sequence, it releases from the template and the RNAS transcript floats away.

RNA Processing

Slicesome cuts out the intron sequences and joins the exons to make the final mRNA

Poly A tails are added to the 3’ end

Methylated G cap added to the 5’ end

More essential terms Intron: region on a gene that is transcribed

into a mRNA molecule but not expressed in a protein; spacer DNA

Exon: region of a gene that directly codes for a protein, it is the region of the gene that is expressed

Eukaryote

Some essential terms

Operon: section of prokaryotic DNA consisting of one or more genes and their controlling elements.

Promoter: the region at the beginning of a gene where RNA polymerase binds; the promoter promotes the recruitment of RNA polymerase and other factors required for transformation

Operator: region on an operon that can either turn on or off expression of a set of genes depending on the binding of a regulatory molecule

Genetic engineers use promoter and operator regions to turn on / off the production of certain genes

Lac Operon

Codon Chart

Translation

Translation Initiation

Initiator tRNA binds to the AUG codon of the mRNA. This tRNA has an anticodon of UAC and carries Met amino acid so all translated products start with the Met amino acid

Translation Elongation

A site – tRNA enters with its new amino acid

P site – growing amino acid chain is linked to newly arriving amino acid by a peptide bond

E site – tRNA leaves without its amino acid

Translation Termination

When stop codon appears in A site, there is no tRNA to bind so a release factor binds instead

This causes the polypeptide chain to release

Finally, it also causes the ribosome subunits to disassociate and translation is terminated

Amino Acids and Proteins

Mutations

• Occur when DNA Polymerase makes a mistake or environmental factors cause an alteration in the DNA sequence

• Mutations can be harmful, beneficial, or neutral