Chapter 12: Molecular Biology of the Gene (Outline) DNA Structure

Watson and Crick Model DNA Replication

Semiconservative Replication Prokaryotic versus Eukaryotic Replication

Types of RNA Gene Expression The Genetic Code Transcription Translation

Structure of DNA

DNA contains Two Nucleotides with purine bases (double ring)

Adenine (A) Guanine (G)

Two Nucleotides with pyrimidine bases (single ring) Thymine (T) Cytosine (C)

Each nucleotide consists of Deoxyribose (5-carbon sugar) Phosphate group A nitrogen-containing base

Chargaff’s Rules

In 1947, Erwin Chargaff had developed a series of rules based on a survey of DNA composition in organisms

The amounts of A, T, G, and C in DNA varies from species to species

In each species, the amount of A=T and the amount of G=C

All this suggests DNA uses complementary base pairing to store genetic info

Rosalind Franklin’s Work

Was an expert in X-ray crystallography Technique used to examine DNA

fibers (under right conditions form a crystal) Concluded that DNA is a double helix

Watson and Crick Model

Watson and Crick, 1953 Constructed a model of DNA from Franklin’s

X-ray diffraction Double-helix model is similar to a twisted ladder

Sugar-phosphate backbones make up the sides Hydrogen-bonded bases make up the rungs

‘steps’

Model agreed with Chargaff’s rule or complementary base pairing

Received a Nobel Prize in 1962

Watson/Crick Model of DNA

Watson and Crick Model (cont.)

Antiparallel nature: the sugar-phosphate backbone of each strand runs in opposite directions

One strand runs 5’ to 3’, while the other runs 3’ to 5’

The nucleotides connect at the hydroxyl group of the 5 carbon sugar (at the 3’ end)

DNA strand is made in a 5’ to 3’ direction

Replication of DNA DNA replication: the process of copying a DNA

molecule Each old DNA strand serves as a template Replication involves 3 main steps

Unwinding – original double helix strands (parental DNA) are unwound and “unziped” by helicase enzyme

Complementary base pairing – positioning of new complementary nucleotides

Joining – complementary nucleotides join to form new strands

Each daughter DNA contains an old & new strand

Semiconservative Replication of DNA

DNA polymerase: enzyme complex that carries out the last two steps in DNA synthesis

DNA replication must occur before cellular division

Cancer cells are treated with chemotherapeutic drugs “analogs”, which causes replication to stop and cells to die off

Replication:Prokaryotic vs. Eukaryotic

Prokaryotic Replication Bacteria have a single circular loop of DNA

Replication moves around the circular DNA molecule in both directions

The process begins at the origin of replication and always occur in the 5’ to 3’ direction

Replication stops when the 2 DNA polymerases meet at a termination region

Bacterial cells require about 40 min to replicate the complete chromosome

Prokaryotic DNA Replication

Replication:Prokaryotic vs. Eukaryotic

Eukaryotic Replication DNA replication begins at numerous points

(origins of replication) along linear chromosome DNA unwinds and unzips into two strands

through the action of helicase enzyme Each old strand of DNA serves as a template for

a new strand Replication bubbles spread bi-directionally until

they meet Replication fork – V shape formed during DNA

replication

Eukaryotic DNA Replication

Eukaryotic DNA Replication (cont.) Eukaryotes replicate their DNA at a slower rate

500–5,000 base pair per minute Eukaryotic cells, however complete DNA

replication in a matter of hours, how? The linear chromosomes also pose a problem:

DNA polymerase cannot replicate the ends of chromosomes that contain telomeres (short segments of DNA repeated over and over)

Instead, telomerase enzymes add the repeats after chromosome replication

In stem cells, this process preserves the ends of chromosomes and prevents the loss of DNA

Accuracy of Replication DNA polymerase is very accurate with approx.

one mistake per 100,000 base pairs DNA polymerase is also capable of proof

reading the daughter strand It recognizes a mismatched nucleotide and

removes it from a daughter strand, how? By reversing direction and removing several

nucleotides

After removing the mismatched nucleotide, it changes direction again and continues

Overall, the error rate for the bacterial DNA polymerase is only one in 100 million base pairs!

The Genetic Code of Life

The mechanism of gene expression Gene – segment of DNA that specify

information, but information is not structure and function (i.e. protein)

Genetic info is expressed into structure and function through protein synthesis

DNA in gene controls the sequence of nucleotides in an RNA molecule

RNA controls the primary structure of a protein

RNA Carries the Information

Like DNA, RNA is a polymer of nucleotides RNA nucleotides are of four types: U, A, C & G Uracil (U) replaces thymine (T) of DNA There are three major classes of RNA

Messenger RNA (mRNA) - takes a message from DNA in the nucleus to ribosomes in cytoplasm

Transfer RNA (tRNA) – transfers amino acids to the ribosomes

Ribosomal RNA (rRNA) – and proteins make up ribosomes which read the message in mRNA

Structure of RNA

The Genetic Code

The central dogma of molecular biology states that the flow of genetic information is “DNA to RNA to protein”

There must be a genetic code for each of the 20 amino acids found in proteins

However, can four nucleotides provide enough combinations to code for 20 amino acids?

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, why?

Central Dogma in Molecular Biology

Transcription: DNA serves as a template for RNA formation

Translation: mRNA transcript directs the amino acid sequence in a polypeptide

Finding the Genetic Code

Nirenberg and Matthei (1961) found that an enzyme that could be used to construct a synthetic RNA in a cell-free system; they showed the codon UUU coded for phenylalanine

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

Properties of the Genetic Code

The code is degenerate There are 64 codons available for 20 amino

acids Most amino acids encoded by two or more

codons (e.g. luecine and serine), why? The genetic code is unambiguous

Each triplet codon specifies one and only one amino acid

The code has start and stop signals There are one start codon and three stop

codons (sequences)

The Code is Universal With few exceptions, all organisms use the

code the same way Genetic code used by mammalian mitochondria

and chloroplasts differs slightly

The universal nature of the genetic code suggests the code dates back to the very first organisms and that all organisms are related

It is possible to transfer genes from one organism to another – genetic engineering Example: Glowing mice

mRNA Codons

Steps in Gene Expression:(1) Transcription

Messenger RNA is formed A DNA segment helix unwinds and unzips,

thus serving as a template for mRNA formation

Loose RNA nucleotides bind to exposed DNA bases using the C=G AND A=U rule

The information is in the base sequence of the “template” strand of the DNA molecule

RNA polymerase connects the loose RNA nucleotides together in the 5’ → 3’ direction

Transcription of mRNA

Transcription (initiation) begins when RNA polymerase attaches to a promoter on DNA

Promoter – region of DNA which defines the start of the gene, the direction of transcription, and the strand to be transcribed

The RNA-DNA association is not as stable as the DNA double helix

Only the newest portion of the RNA molecule with RNA polymerase is bound to DNA; the rest dangles off to the side

Transcription of mRNA (cont.)

Elongation of mRNA continues until RNA polymerase comes to a DNA stop sequence

Results in the release the mRNA transcript

Many RNA polymerase molecules work to produce mRNA from the same DNA region at the same time

Either strand of DNA can be a template strand but for a different gene

Transcription

Steps in Gene Expression:(2) Translation

Translation takes place in the cytoplasm of eukaryotic cells

Translation is the second step by which gene expression leads to protein synthesis

The sequence of codons in the mRNA at a ribosome directs the sequence of amino acids in a polypeptide

One language (nucleic acids) is translated into another language (protein)

The Role of Transfer RNA

The tRNA molecule transfers amino acids to the ribosomes

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

There is at least one tRNA molecule for each of the 20 amino acids found in proteins

There are fewer tRNAs (40) than codons (61) as some tRNAs pair with more than one codon

Structure of tRNA

Translation Requires Three Steps

During translation, mRNA codons base-pair with tRNA anticodons carrying specific amino acids

Codon order determines the order of tRNA molecules and the sequence of amino acids in polypeptides

Protein synthesis involves 3 steps: initiation, elongation, and termination

Enzymes are required for all three steps; energy (ATP) is needed for the first two steps

Steps in Translation:1. Initiation Components necessary for initiation are

Small ribosomal subunit mRNA transcript Initiator tRNA, and Large ribosomal subunit Initiation factors - special proteins that bring the

above together Initiator tRNA

Always has the UAC anticodon Always carries the amino acid methionine Capable of binding to the P site of ribosome

Steps in Translation:1. Initiation (cont.)

Chain Initiation In prokaryotes, a small ribosomal subunit attaches to

mRNA at the start codon (AUG)

Initiator tRNA (UAC) pairs with this codon; then the large ribosomal subunit joins to the small subunit

Each ribosome contains three binding sites – the P site (for peptide), the A site (for amino acid), and the E site (for exit)

The initiator tRNA binds to the P site

The A site is for the next tRNA carrying the next aa

The E site is to discharge tRNAs from the ribosome

Initiation

Steps in Translation:2. Elongation

Elongation – refers to the growth in length of the polypeptide one amino acid at a time

The tRNA with attached polypeptide is at the P site; a tRNA-amino acid complex arrives at the A site

Elongation factors – proteins that facilitate complementary base pairing between the tRNA anticodon and the mRNA codon at the ribosome

The polypeptide is transferred and attached by a peptide bond to the newly arrived amino acid in the A site via a ribozyme and energy (ATP)

Steps in Translation:2. Elongation (cont.)

The tRNA molecule in the P site is now empty 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 → exits the ribosome

As the ribosome moves forward three nucleotides, there is a new codon now located at the empty A site

The complete cycle is rapidly repeated, about 15 times per second in the bacterium E. coli

Elongation

Steps in Translation:3. Termination

Termination of polypeptide synthesis occurs at a stop codon UAA, UAG, or UGA Does not code for an amino acid

The polypeptide is enzymatically cleaved from the last tRNA by a release factor

The tRNA and polypeptide leave the ribosome, which dissociates into its two subunits

The released polypeptide begins to take on its 3D shape

Termination