Gene Expression Basics

Studying Genetics in the Twenty-First Century
CHAPTER
Genetics: The study of biological information
COURSE OUTLINE
1. Genes: Fundamental Informational Molecules of Life 2. Eukaryotic Gene regulation: Functional Molecules of Life Processes 3. Modern Genetic Techniques 4. Complex Systems and Molecular Interactions in cancer genetics
PART I
What Genes Are and What They Do
CHAPTER
Gene Expression: The Flow of Information from DNA to RNA to Protein
OUTLINE
1.1 The Genetic Code information to functional molecules 1.2 Transcription: From DNA to RNA 1.3 Translation: From mRNA to Protein 1.4 The Effect of Mutations on Gene Expression and Gene Function
Three levels of biological information

DNA Macromolecule made of nucleic acids Repository of the genetic code Proteins Macromolecules made of amino acids Amino acid sequence determined by DNA sequence Biological systems Network of interactions between molecules or groups of cells Accomplish coordinated functions
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display 3 Hartwell et al., 4th ed., Chapter 1
Where and how is biological information stored

Biological function
= protein expression
=
gene expression
Appreciating the complexity of biological information

Double stranded DNA Hydrogen bonds Complementary base pairs composed of sugar, amine bonds, phosphate groups Conversion of a 3-D functional molecule to a 1-D molecule for computational analysis
Complementary base pairs are a key feature of the DNA molecule

DNA is comprised of four nitrogenous bases [guanine (G), adenine (A), cytosine (C), and thymine (T)], a deoxyribose, and a phosphate
G C and A T hydrogen bonds between each strand of the double helix
The two strands of the double helix are in opposite orientation
Organization of genetic information in cells

Genes are sequences of DNA that encode proteins Chromosomes are organelles that package and manage the storage, duplication, expression, and evolution of DNA
Genomes are the entire collection of chromosomes in each cell of an organism

The human genome: 24 kinds of chromosomes 3 x 109 base pairs Encodes 20,000 30,000 genes
Proteins are polymers of hundreds to thousands of amino acids

There are 20 different amino acids Information in DNA of a gene dictates the sequence of amino acids for the protein The order of amino acids determines the type of protein and its three dimensional structure Diversity of three-dimensional structure of protein generates an extraordinary diversity of protein function
The amino acid sequence determines the three-dimensional shape of the protein
Chemical formulas for two amino acids Three-dimensional shapes of two proteins
RNA evolved into an intermediary in conversion of DNA information into protein

RNA is comprised of four nitrogenous bases [guanine (G), adenine (A), cytosine (C), and uracil (U)], a ribose, and a phosphate
Bases are read as triplets to encode amino acid subunits of protein
All living organisms use essentially the same genetic code
Specific triplets of RNA bases encode the 20 amino acids
Four general themes for gene expression

Pairing of complementary bases is the key to the transfer of information from DNA to RNA and from RNA to protein Polarities of DNA, RNA, and polypeptides help guide the mechanisms of gene expression Gene expression requires input of energy and participation of specific proteins and macromolecular assemblies Mutations that change genetic information or obstruct the flow of its expression can have dramatic effects on phenotype
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8
12
Mutation example in Predictive and Preventive Medicine

Genetic variations can cause or predispose
Single DNA base pair mutation in the betaglobin gene will lead to sickle cell anemia Mutation in the breast cancer BRCA1 gene results in a 70% chance in acquiring the disease
Differences: control of genetic expression
Gene expression: the flow of genetic information from DNA via RNA to protein
RNA polymerase transcribes DNA to produce an RNA transcript
Ribosomes translate the mRNA sequence to synthesize a polypeptide
Translation follows the "genetic code"

14
Summary of the genetic code

Genetic code has triplet codons
Codons are nonoverlapping Three nonsense codons dont encode an amino acid; UAA (ocher), UAG (amber) and UGA (opal) Genetic code is degenerate Reading frame is established from a fixed starting point codon for translation initiation is AUG mRNAs and polypeptides have corresponding polarities Mutations can be created in three ways; frameshift, missense, and nonsense
Copyright The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8 15
Transcription: From DNA to RNA

RNA polymerase catalyzes transcription
Promoters are DNA sequences that provide the signal to RNA polymerase for starting transcription
RNA polymerase adds nucleotides in 5-to-3 direction
Formation of phosphodiester bonds using ribonucleotide triphosphates (ATP, CTP, GTP, and UTP)
Hydrolysis of bonds in NTPs provides energy for transcription Terminators are RNA sequences that provide the signal to RNA polymerase for stopping transcription
Structure of the methylated cap at the 5' end of eukaryotic mRNAs

Capping enzyme adds a "backward" G to the 1st nucleotide of a primary transcript
Transcribed bases
Methylated cap not transcribed
Triphosphate bridge
17
Processing adds a tail to the 3' end of eukaryotic mRNAs
18
RNA splicing removes introns
Exons sequences found in a genes DNA and mature mRNA (expressed regions)
Introns sequences found in DNA but not in mRNA (intervening regions) Some eukaryotic genes have many introns
19
The human dystrophin gene: An extreme example of RNA splicing

Splicing removes introns from a primary transcript
20
RNA processing splices out introns and joins adjacent exons

Short sequences in the primary transcript dictate where splicing occurs
21
RNA processing splices out introns and joins adjacent exons (cont)
Two sequential cuts remove an intron
22
Splicing is catalyzed by the spliceosome
23
splicing video
http://www.youtube.com/watch?v=FVuAwBGw_pQ
24
Translation: From mRNA to protein

Transfer RNAs (tRNAs) mediate translation of mRNA codons to amino acids Translation takes place on ribosomes that coordinate movement of tRNAs carrying specific amino acids
tRNAs are short single-stranded RNAs of 74 95 nt

Each tRNA has an anticodon that is complementary to an mRNA codon A specific tRNA is covalently coupled to a specific amino acid (charged tRNA) Base pairing between an mRNA codon and an anticodon of a charged tRNA directs amino acid incorporation into a growing polypeptide
Some tRNAs contain modified bases
26
Three levels of tRNA structure

Nucleotide sequence is the primary structure Secondary structure (cloverleaf shape) is formed because of short complementary sequences within the tRNA Tertiary structure (L shape) is formed by 3-dimensional folding
Aminoacyl-tRNA synthetases catalyze attachment of amino acids to specific tRNAs

Each aminoacyl-tRNA synthetase recognizes a specific amino acid and the structural features of its corresponding tRNA
28
Base pairing between an mRNA codon and a tRNA anticodon determines which amino acid is added to a growing polypeptide
Fig. 8.21
29
Wobble: Some tRNAs recognize more than one codon for the amino acid they carry
30
A ribosome has two subunits composed of RNA and protein
31
Mechanism of translation
Initiation stage - start codon is AUG at 5 end of mRNA
In bacteria, initiator tRNA has formylated methionine (fMet)
Elongation stage - amino acids are added to growing polypeptide

Ribosomes move in 5-to-3 direction along mRNA 2-15 amino acids added to C terminus per second
Termination stage - polypeptide synthesis stops at the 3' end of the reading frame
Recognition of nonsense codons Polypeptide synthesis halted by release factors Release of ribosomes, polypeptide, and mRNA
Different parts of a ribosome have different functions

Small subunit binds to mRNA Large subunit has peptidyl transferase activity Three distinct tRNA binding areas E, P, and A sites
33
Translation of mRNAs on ribosomes: Initiation phase in prokaryotes

Ribosome binding site consists of a Shine-Dalgarno sequence and an AUG Three sequential steps: small ribosomal subunit binds first, fMet-tRNA positioned in P site, large subunit binds Initiation factors (not shown) play a transient role
34
Translation of mRNAs on ribosomes: Initiation phase in eukaryotes

Small ribosomal subunit binds to 5' cap, then scans the mRNA for the first AUG codon Initiator tRNA carries Met (not fMet)
35
Translation of mRNAs on ribosomes: Elongation phase

Addition of amino acids to C-terminus of polypeptide
Charged tRNAs ushered into A site by elongation factors (not shown)
36
Polyribosomes consist of several ribosomes translating the same mRNA

Simultaneous synthesis of many copies of a polypeptide from a single mRNA
37
Translation of mRNAs on ribosomes: Termination phase

No normal tRNAs carry anticodons for the stop codons
Release factors bind to the stop codons

Release of ribosomal subunits, mRNA, and polypeptide
38
Posttranslational processing can modify polypeptide structure

(a) Cleavage may remove an amino acid (c) Chemical constituent addition may modify a protein
(b) Cleavage may split a polyprotein
39

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Studying Genetics in the Twenty-First Century

Genetics: The study of biological information

What Genes Are and What They Do

Gene Expression: The Flow of Information from DNA to RNA to Protein

Three levels of biological information

Where and how is biological information stored

Appreciating the complexity of biological information

Complementary base pairs are a key feature of the DNA molecule

The two strands of the double helix are in opposite orientation

Organization of genetic information in cells

Genomes are the entire collection of chromosomes in each cell of an organism

Proteins are polymers of hundreds to thousands of amino acids

RNA evolved into an intermediary in conversion of DNA information into protein

Bases are read as triplets to encode amino acid subunits of protein

All living organisms use essentially the same genetic code

Specific triplets of RNA bases encode the 20 amino acids

Four general themes for gene expression

Mutation example in Predictive and Preventive Medicine

Differences: control of genetic expression

Ribosomes translate the mRNA sequence to synthesize a polypeptide

Translation follows the "genetic code"

Summary of the genetic code

Transcription: From DNA to RNA

Structure of the methylated cap at the 5' end of eukaryotic mRNAs

Methylated cap not transcribed

Processing adds a tail to the 3' end of eukaryotic mRNAs

RNA splicing removes introns

The human dystrophin gene: An extreme example of RNA splicing

RNA processing splices out introns and joins adjacent exons

Splicing is catalyzed by the spliceosome

Translation: From mRNA to protein

tRNAs are short single-stranded RNAs of 74 95 nt

Some tRNAs contain modified bases

Three levels of tRNA structure

Aminoacyl-tRNA synthetases catalyze attachment of amino acids to specific tRNAs

A ribosome has two subunits composed of RNA and protein

Elongation stage - amino acids are added to growing polypeptide

Different parts of a ribosome have different functions

Translation of mRNAs on ribosomes: Initiation phase in prokaryotes

Translation of mRNAs on ribosomes: Initiation phase in eukaryotes

Translation of mRNAs on ribosomes: Elongation phase

Charged tRNAs ushered into A site by elongation factors (not shown)

Polyribosomes consist of several ribosomes translating the same mRNA

Translation of mRNAs on ribosomes: Termination phase

Release factors bind to the stop codons

Posttranslational processing can modify polypeptide structure

(b) Cleavage may split a polyprotein

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