From Gene to Protein Lecture NotesBiol 100 – K.Marr

1. Topics for the next few lectures

– Transcription: From DNA to RNA

– Translation: From RNA to Protein

– Understanding Cystic Fibrosis

– Chapter 10 in Essential Biology by Campbell et al

2. Lab 7. Modeling DNA Structure, DNA Replication and Protein Synthesis Read the introduction carefully

– Part 1 (through page 9)—modeling DNA Structure and Replication

– Part 2—modeling transcription and translation

Cytoplasm

Nucleus

DNA

Transcription

Translation

Protein

mRNA

• Transcription: DNA copied into mRNA molecule

• Translation: ribosomes translate mRNA into protein—a chain of amino acids

• Proteins control phenotype. How?

The Flow of Genetic Information: DNA to RNA to Protein Phenotype

A few of the many roles played by proteins:

1. Enzymes: catalysts for nearly all chemical reactions in cells; Determine what cells can make and digest

2. Structural components: muscles (actin and myosin), connective tissue (collagen, elastin)

3. Receptors on cell surface for growth factors, hormones, etc.

4. Hormones: e.g. insulin, growth hormone, prolactin

5. Transport: e.g. hemoglobin, spindle fibers

6. Immune system: antibodies

The one gene–one protein hypothesis:The function of a gene is to dictate the production of a specific protein. Why are proteins so important?

CF phenotype

• Genes determine which proteins a cell can make

• Proteins control phenotype

• e.g. CFTR Gene codes for CFTR protein

CFTR Protein: The cystic fibrosis transmembrane regulator proteinCarbohydrate

Chloride ions

Water

CFTR Protein

Cellmembrane

Cytoplasm of cell lining duct or lungs

Water

Inside of duct or

Air sac in lungs

CFTR Protein

• Pumps chloride ions (salt) into cells lining ducts or the lungs

• What are the consequences when CFTR doesn’t work?

• How does a gene control the production of a protein?

The order of Bases in a gene determines the order of amino acids in the protein it codes for

Is the order of amino acids in a protein important?

• View animation of transcription

Questions to answer:

1. What do we start with and end with?

2. Where does transcription occur? When?

3. What is needed for transcription to occur?

4. What is the sequence of events?

Transcription: copying DNA into RNA

An RNA Nucleotide

Phosphate

Sugar:

ribose

This oxygen isabsent in deoxyribose

Base

(Uracil, U)

RNApolymerase

RNA nucleotides

Newly madeRNA Direction of

transcriptionTemplatestrand of DNA

Transcription of a gene by RNA polymerase

(a) Parent DNA

Strandseparation

(b) Transcription begins

RNApolymerase

Complementarybase pairing

Transcription: copying DNA into RNA ( 1 of 2)

(c) Transcription continues (d) Products of transcription

Non-codingstrand

Codingstrand

Nucleotidejoining

New RNA strand(actually severalhundred basepairs long)

Parent DNAtotallyconserved

Transcription: copying DNA into RNA ( 2 of 2)

Comparing DNA and RNA

DNA RNA

Number of Strands

Sugars

Bases

Fig. 7.07

(a) Gene

(b) Primary transcriptTranscription

RNA splicing:

Differential splicing can result in different mRNA molecules and, therefore, different proteins

RNA Processing

Translation

(c) Spliced RNA

(d) Mature RNA

(d) protein

Exon 1 Exon 2

Intron 1

Exon 3

Intron 2

Exon 4

Intron 3

Exon 5

Intron 4

Exon 6

Intron 5

Transcription in Eukaryotic Cells: Differential RNA splicing can result in one gene producing more than one protein

Processing of Eukaryotic RNA

RNA Processing includes• Adding a cap and tail

• Removing introns

• Splicing exons together

– Differential splicing produces different mRNA molecules

Gene (DNA)

RNAtranscriptwith capand tail

mRNA

Exon Intron ExonIntron Exon

Cap

Introns removed Tail

Exons spliced together

Coding sequence

Nucleus

Cytoplasm

Transcription + theAddition of cap and tail

• View animation of translation

Questions to answer1. What do we start with and end with?

2. Where does translation occur?

3. What is needed for translation to occur?

4. What is the sequence of events?

5. What are the roles of mRNA, ribosomes, start codon, tRNA, anticodons, stop codon?

Translation: Ribosomes reading mRNA to produce a polypeptide

Transfer RNA: tRNA

tRNA1. Acts as a

molecular interpreter

2. Carries amino acids

3. Matches amino acids with codons in mRNA using anticodons

Amino acid attachment site

Hydrogen bond

RNA polynucleotide chain

AnticodonAnticodon

Amino acid

Codon on mRNAmRNA

A portion of an mRNA molecule attached to a tRNA

Each Codon codes

Specifies a specific

tRNA—amino acid

complex

Proteinunderconstruction

Largesubunit

Smallsubunit

A ribosome translating mRNA into protein

mRNA

Ribosomes• Organelle that makes

protein

• Reads mRNA 5’ 3’

• Made of rRNA and protein

• Consist of 2 subunits

1. Initiation of Translation

Anticodon

tRNA

Amino acid

Ribosome

mRNA

Codon

2. Elongation

Peptide bond forms

2. Elongation continues: Translocation of Ribosome

tRNA ejected

Ribosomemoves

3. Termination of Translation

tRNA ejected

Ribosomemoves

Terminationfactor binds

Peptide bond forms

3. Termination continued: Disassembly of Ribosome

Polypeptide chain

tRNA

Transcription & Translation of the CRTR Gene in Healthy People

Part of a normal CFTR gene:

5’...ATCATCTTTGGTGTT...3’ non-coding strand

3’...TAGTAGAAACCACAA...5’ coding strand

1. Transcribe this portion of the gene.

The whole gene codes for 1480 amino acids in CFTR protein!

What is the order of bases in the resulting mRNA molecule?

2. Translate this portion of the gene.

– What is the order of amino acids in the resulting protein?

Table of Codons

found on

mRNA

• Each codon specifies a specific amino acid

• The same genetic code is used by nearly all organisms!!

Part of a normal CFTR gene:

5’...ATCATCTTTGGTGTT...3’ non-coding strand

3’...TAGTAGAAACCACAA...5’ coding strand

5’...AUCAUCUUUGGUGUU...3’

Transcription

.....Ile-Ile-Phe-Gly-Val…

(only 5 of the 1480 amino acids in protein!!)

Translation

Transcription & Translation of the CRTR Gene in Healthy People

Part of CFTR gene associated with Cystic Fibrosis:

5’...ATCATTGGTGTT...3’ non-coding strand

3’...TAGTAACCACAA...5’ coding strand

1. Transcribe this portion of the gene.

What is the order of bases in the resulting mRNA molecule?

2. Translate this portion of the gene.

– What is the order of amino acids in the resulting protein?

3. What is different about the gene and the protein in people with cystic fibrosis?

Transcription & Translation of the CRTR Gene in People with CF

Part of CFTR gene associated with Cystic Fibrosis:

5’...ATCATTGGTGTT...3’ non-coding strand

3’...TAGTAACCACAA...5’ coding strandTranscription

Translation

Transcription & Translation of the CRTR Gene in People with CF

5’...AUCAUUGGUGUU...3’

.....Ile-Ile-Gly-Val…….. Phenylalanine (Phe) is missing

Explaining the symptoms of CF

• Why does CF only affect certain parts of the body?

• What do the characteristics of CF have in common?

1. Mucus build-up in the lungs

• Lung infections (e.g. pneumonia)

2. Male sterility (blocked vas deferens)

3. Salty sweat

4. Trouble digesting food (blocked pancreatic duct)

Explaining the symptoms of CF

Chloride ions in cell

Chloride ions outside of cell CFTR Protein: Pumps

Chloride ions into cell

• In CF, the faulty CFTR protein never makes it to cell membrane

1. What builds up outside of cells? Why?

2. Why salty sweat?

3. Why does mucus collect in lungs?

4. Why respiratory infections?

5. Why problems with digestion?

6. Why male sterility?

Understanding Cystic Fibrosis at the Cellular Level

How does CFTR protein get from where it’s produced to its home in the cell membrane?

1. Where is the CFTR protein produced?

2. CFTR is a glycoprotein—where does it go for modification?

How does it get there?

3. How does the modified CFTR protein get to the plasma membrane?

4. The defective CFTR protein is recognized at the ER as defective

Where is the defective CFTR protein sent?

CF symptoms may be mild or severe

Several hundred different mutations are associated with CF

CFTR Gene

What’s a Mutation?• Any change in the nucleotide sequence of DNA

• Types of Mutations

– Substitution, insertion or deletion

– Occur during DNA replication

• Mutations may Result from:

– Errors in DNA replication

– Mutagens

• physical or chemical agents that may cause errors during DNA replication

chemicals in cigarette smoke

Radiation (e.g. U.V. light, X-rays)

• Why does isoleucine (Ile) at amino acid position 507 remain unchanged?

F508 deletion: the most common cause of cystic fibrosis

Mutations responsible for Sickle Cell Anemia

Normal hemoglobin Sickle-cell hemoglobin

Glu Val

Normal hemoglobin DNA Mutant hemoglobin DNA

mRNA mRNA

• Only one amino acid in 146 is incorrect in sickle-cell hemoglobin!

Types of Mutations: Base Substitutions, Insertions or deletions

• Base substitutions

– May result in changes in the amino acid sequence in a protein, or

– May be silent (have no effect)

mRNA

Protein Met Lys Phe Gly Ala

(a) Base substitution

Met Lys Phe Ser Ala

Types of Mutations: Base Insertions and deletions

• Can have disastrous effects

– Change the reading frame of the genetic message

Met Lys Leu Ala His

(b) Nucleotide deletion

mRNA

Protein Met Lys Phe Gly Ala

• Although mutations are often harmful

– They are the source of the rich diversity of genes in the living world

– They contribute to the process of evolution by natural selection

DNA and RNA: Polymers of Nucleotides

SUMMARY OF KEY CONCEPTS

DNA

Polynucleotide

Nitrogenousbase

Phosphategroup

Nucleotide

Sugar

1

2

4

3. Amino acid attachment

55. Elongation

4. Initiation of translation

6. Termination

1. Transcription

2. RNA processing

RNA Polymerase

Nucleus

DNA

RNA transcript

Intron

Tail

Intron

mRNA

CAP

tRN

A

Enzyme

Amino acid

Ribosomal subunits

Anticodon

Codon

Stop codon

Review: DNA RNA Protein