Gene ActivityGene Activity 1

Ch. 12-b Outline – Gene ActivityCh. 12-b Outline – Gene Activity

Gene ActivityGene Activity 2

Function of Genes

Archibald Garrod:

● First to propose a link between genes and proteins and metabolic diseases.

● Inherited disorders could be caused by lack of a particular enzyme in a metabolic

pathway.

Gene ActivityGene Activity 3

Genes Specify Enzymes

Beadle and Tatum:

Experiments on fungus Neurospora crassa

Induced mutations with X-rays. Spores were no longer able to grow on minimal media.

Figured out which enzymes were lacking & found that each mutant strain only had 1 defective gene

Proposed that each gene specifies the synthesis of one enzyme

One-gene-one-enzyme hypothesis

4Beadle & Tatum Experiment

Gene ActivityGene Activity 5

Genes Specify a Polypeptide

A gene is a segment of DNA that specifies the sequence of amino acids in a polypeptide

Suggests that genetic mutations cause changes in the primary structure of a protein

Examples of genetic mutations that cause disease:

1. Sickle cell disease : a change in DNA causes hemoglobin to have one different amino acid which changes the function of the entire protein. (This was discovered by Linus Pauling and Harvey Itano in 1949)

6Sickle-Cell Disease in Humans

Gene ActivityGene Activity 7Protein Synthesis:From DNA to RNA to Protein

The mechanism of gene expression

DNA in genes specify information, but information is not structure and function

Genetic info is expressed into structure & function through protein synthesis

The expression of genetic info into structure & function:

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

RNA controls the primary structure of a protein

Gene ActivityGene Activity 8

Types of RNA (see transparency here)

RNA is a polymer of RNA nucleotides

RNA nucleotides are of four types:

Uracil, Adenine, Cytosine, and Guanine

Uracil (U) replaces thymine (T) of DNA

Types of RNA Messenger (mRNA) - Takes genetic message from DNA in nucleus to ribosomes in cytoplasm

Ribosomal (rRNA) - Makes up ribosomes which read the message in mRNA

Transfer (tRNA) - Transfers appropriate amino acid to ribosome when “instructed”

9Structure of RNA

10Three Types of RNA

Gene ActivityGene Activity 11Steps in Gene Expression:

There are two steps in gene expression:

1. Transcription

● DNA serves as a template for RNA formation

2. Translation

● mRNA transcript directs the creation of a sequence of amino acids in a polypeptide

12Overview of Gene Expression

Gene ActivityGene Activity 13

The Genetic Code

Each of the 20 amino acids found in proteins is specified by one or more codons Genetic alphabet has only four “letters”: U,A,C,G

Codons in the genetic code are all three bases long

There are 64 possible arrangements of four symbols taken three at a timeOften referred to as triplets

Genetic language has 64 “words” that code for 20 amino acids

Gene ActivityGene Activity 14

Finding the Genetic Code

Nirenberg & Matthei (1961)1. They found that cell enzymes could be used

to construct synthetic RNA.2. Then found that these RNA could be

translated in a test tube into polypeptides3. The first synthetic RNA was made of only U.

● This resulted in a polypeptide made up only of phenylalanine.

4. They repeated this with all possible triplet combinations until they constructed the genetic code table.

15The Genetic Code (mRNA)

CAG

Gene ActivityGene Activity 16

Genetic Code is Universal

All organisms use the same genetic code- Slight differences in mitochondria and chloroplasts DNA, however.

Degenerate (redundant) There are 64 codons available for 20 amino acids

Most amino acids encoded by two or more codons

Contains start and stop signals Makes it possible to transfer genes between different organisms.

Gene ActivityGene Activity 17Steps in Gene Expression:First = Transcription

All types of RNA are created by transcription

DNA unzips and exposes unpaired bases

One side serves as template for mRNA formation. This strand is also called the sense strand.

Loose RNA nucleotides bind to template DNA bases using the C=G & A=U rule

This occurs with the help of RNA polymerase.

● The strand of DNA that is not transcribed is called the noncoding strand or nonsense strand.

Gene ActivityGene Activity 18Steps in Gene Expression:Transcription (cont’d)

Transcription begins when RNA polymerase attaches to a region of DNA known as the promoter.

RNA polymerase joins the nucleotides together in the 5’ to 3’ direction.

- It only adds a nucleotide to the 3’ end of the RNA being formed.

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

Gene ActivityGene Activity 19Steps in Gene Expression:Transcription (cont’d)

When entire gene is transcribed into mRNA, result is an mRNA transcript of the gene

The base sequence in the mRNA is complementary to the base sequence in DNA

Many RNA polymerase molecules can be working at the same time. Thus, a cell can produce thousands of copies of the same mRNA within a short period of time.

20Transcription

21RNA Polymerase

Gene ActivityGene Activity 22

Processing Messenger RNA

In eukaryotes the primary mRNA transcript is modified before it leaves the nucleusRNA splicing occurs:

Primary transcript consists of:Some segments that will not be expressed (introns)

Segments that will be expressed (exons) Performed by spliceosome complexes in nucleoplasmIntrons are excised (cut out)Remaining exons are spliced back together

Gene ActivityGene Activity 23

Processing Messenger RNA (cont’d) Modifications to ends of primary transcript:

Cap of modified guanine on 5′ end Poly-A tail of 150+ adenines on 3′ end

Result is mature mRNA transcript

24

mRNA Processing

in Eukaryotes

Gene ActivityGene Activity 25

Functions of Introns

Eukaryotes have many introns while prokaryotes don’t.In humans up to 95% of genes are introns.

Genome has only about 25,000 coding genes

Possible functions of introns: Exons might combine in various combinations

Would allow different mRNAs to result from one segment of DNA

Introns might regulate gene expression

Gene ActivityGene Activity 26Steps in Gene Expression:Second = Translation

● Takes place in cytoplasm.

● Codons in mRNA direct the sequence of amino acids in a polypeptide. One “language” is translated into another.

● Involves all three types of RNA

● tRNA

- Single-stranded RNA

- Transfers amino acids to the ribosomes.

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

Gene ActivityGene Activity 27Steps in Gene Expression:Role of tRNA

tRNA molecules have two binding sites: An amino acid binds to the 3’ end of the tRNA

- This is helped along by an enzyme called aminoacyl-tRNA synthetase.

The opposite end of the tRNA contains an anticodon:

- This is a group of 3 bases that are complementary to a specific codon of mRNA

All tRNA molecules with a specific anticodon will always bind with the same amino acid

28Structure of tRNA

Gene ActivityGene Activity 29

Role of Ribosomes

Ribosomal RNA (rRNA):Produced from a DNA template in the nucleolus

Combined with proteins into large and small ribosomal subunits

A completed ribosome has three binding sites to facilitate pairing between tRNA and mRNAThe E (for exit) site

The P (for peptide) site, and

The A (for amino acid) site

30Ribosomal Binding Sites

31Ribosomal Structure and Function

Gene ActivityGene Activity 32Steps in Gene Expression:Second = Translation

An mRNA transcript migrates to rough endoplasmic reticulum

Associates with the rRNA of a ribosome

The ribosome “reads” the information in the transcript

Ribosome directs various species of tRNA to bring in their specific amino acid “fares”

tRNA specified is determined by the code being translated in the codons of mRNA transcript

Gene ActivityGene Activity 33Steps in Translation:#1 - Initiation

Components necessary for initiation are:Small & large ribosomal subunitsmRNA transcript Initiator tRNA Initiation factors (special proteins that bring the above together)

Initiator tRNA:Always has the UAC anticodonAlways carries the amino acid methionineCapable of binding to the P site

Gene ActivityGene Activity 34Steps in Translation:#1 - Initiation

● Small ribosomal subunit attaches to mRNA transcript

● Initiator tRNA (UAC) attaches to P site

- Beginning of transcript always has the START codon (AUG). So first tRNA always carries the amino acid methione.

● Large ribosomal subunit joins the small subunit

35

Steps in Translation:

#1 - Initiation

Gene ActivityGene Activity 36Steps in Translation:#2 - Elongation

“Elongation” refers to the growth in length of the polypeptide

● tRNA molecules bring their amino acid fares to the ribosome

They must have the anticodon complementary to the mRNA codon

being read

Joins the ribosome at it’s A site

Methionine of initiator is connected to amino acid of 2nd tRNA by peptide bond

Gene ActivityGene Activity 37Steps in Translation:#2 – Elongation (cont’d)

Second tRNA moves over to the P site (translocation)

Spent initiator moves to E site and exits

Ribosome reads the next codon in the mRNA

Joins the ribosome at it’s A site

Dipeptide on 2nd amino acid is connected to amino acid of 3rd tRNA by peptide bond

- A ribozyme and energy is needed to bring about this transfer.

38Steps in Translation:# 2 Elongation

Gene ActivityGene Activity 39Steps in Translation:#3 – Termination

Final step in protein synthesis:● Ribosome reads the STOP codon at the end of

the mRNA. These are the following codons:UAA, UAG, or UGAThese do not code for an amino acid

Polypeptide is released from last tRNA by release factor

Ribosome releases mRNA and dissociates into subunits

mRNA read by another ribosome

40Steps in Translation:

#3 - Termination

41Summary of Gene Expression

(Eukaryotes)

42DNA Replication Video I

http://www.courses.fas.harvard.edu/~biotext/animations/replication1.html

DNA Replication Video II

http://highered.mcgraw-hill.com/olc/dl/120076/bio23.swf

Protein Synthesis Video

http://highered.mcgraw-hill.com/olc/dl/120077/micro06.swf