Protein Synthesis: From Gene to

Protein

Step #1 DNA unwinds and exposes the bases (Initiation)Begins at the Promoter Site

1. TATA Box (25 bases upstream)a. Area that signals the RNA Polymerase II to attach to the DNAb. Transcription Factors bind to the TATA Box to help the RNA

Polymerase II to attach to the DNA2. Transcription Initiation Complex: Transcription Factors + RNA Polymerase at the promoter site

ATATTTTTATAAAA

TATA BOX

Promoter Site

Template DNA (Gene)

Initial Transcription Factor

Transcription Factors

RNA Polymerase II

Transcription Initiation Complex

Why is this important?

How is a gene found from 3 billion base pairs?

17-06-Transcription.mov

Step #2 mRNA made from DNA (Transcription)

Initiation: Covered in Step One

Elongation: RNA Polymerase moves downstream making RNA in the 5’ 3’ direction of the template strand

Termination: RNA Polymerase released when a terminator sequence is reached

(AATAAA)

Promoter region

Transcription unit (gene)

mRNA

Termination Sequence

mRNA

RNA Polymerase

RNA Polymerase17-06-Transcription.mov

Coding strand

Step #3 mRNA Leaves Nucleus and Goes to the Ribosome

mRNA is modified before it leaves the nucleus

Pre-mRNA

Termination SequenceStop CodonStart Codon

Addition of the 5’ Cap

1. Prevents degradation by enzymes

2. Allows attachment to ribosome

Addition of the Poly (A) tail

1. Prevents degradation by enzymes

2. Facilitates export from the nucleus

Leader TrailerCoding Segment

1. Alteration at the ends of the pre-mRNA

A. RNA Processing:

Processing

mRNA used in Translation

2. Alteration of the coding sequence - mRNA Splicing

5’ Cap

5’ Cap

Poly (A) tail

Poly (A) tail

Pre-mRNA

mRNA

ExonExon ExonIntron Intron

Coding Segment

Enzymes

Part of the RNA that remains to be translated into proteinExon

Intron Part of the RNA that is removed

Intron

5’ Cap

Coding Segment

Exon 1 + Exon 2 + Exon 3 Poly (A) tail

RNA Processing Vocabulary

Exon ExonIntron

Removed Intron

mRNA

Proteins

snRNP’s

Spliceosome

snRNA

Pre-RNA

Spliceosome Components

snRNA’sProteins

snRNP’s

Spliceosome

Ribozymes

What are the Functions of introns and exons?

1. Allows one code to make different proteins from generic “mix and match” domains

2. Controls gene expression

Small Nuclear RNA

‘SNURPS” Small Ribonucleoproteins

The combination of snRNA + snRNP + Protein used to cut out introns

RNA that function as enzymes. Some RNA’s can splice themselves

B. Attachment to the Ribosome1. Ribosome Anatomy

Small Subunit

Large Subunit

E Site

P site

A Site

mRNA binding site

2. Steps of Attachment (Initiation)

a) mRNA attaches to the smaller unit

b) Initiator tRNA with anticodon UAC bonds to start codon AUG

U A C A U G Met

c) Large ribosomal unit joins small unit with the tRNA at the P site.

d) GTP provides the energy

GTP

Met

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Step #4 tRNA Bonds to Appropriate Amino Acid

Requirements

1. Aminoacyl-tRNA Synthase enzymeAminoacyl-tRNA Synthase

Specific Amino Acid

2. Specific Amino Acid

3. ATP

4. Specific tRNA

Active site fits specific Amino Acid

Active Site specific for anticodon

tRNA

Aminoacyl tRNA

“activated amino acid”

Step # 5 tRNA bonds to mRNA at rRNA

Step # 6 Amino Acids Bond to make the ProteinAll part of Translation

Steps in Translation1. Initiation – attachment of mRNA to the ribosome

(This was already covered in Step # 3)

2. Elongation – the addition of amino acids to the growing protein chain

a) Codon Recognition : tRNA binds to codon at A site

GTP

GDP

GTP

GDP

b) Peptide Bond Formation: Amino acids bond to form protein chainc) Translocation:

tRNA in P site is shifted to the E site

E Site

P site

A Site

Note: Growing Chain

3. Termination: Completed Protein and mRNA released from the Ribosome

Stop Codons

(UAG, UAA, UGA)

Release Factor Protein

a) Release factor protein binds at stop codon at the A site

b) The release factor breaks bond of the tRNA and the last amino acid of the ribosome releasing the tRNA

c) The 2 ribosomal units, mRNA, release factor dissociate

17-17-Translation.mov

Step #7 tRNA goes back to pick up more amino acids;

mRNA breaks down;

Funtional protein ready for use

A. mRNA

2. Many proteins can be made with one mRNA transcript

1. May not immediately break down but bond to another ribosome to form a polyribosome

B. Protein Most proteins must be modified to be functional

1. Chaperonins needed to fold protein into correct tertiary structure

2. Secretory proteins are targeted to be modified in the Rough ER prior to export

-amino acids cleaved (methionine), functional groups added, tertiary and quaternary structures modified

Reading the Code3’ – TACGGCCGATTCTGACATCGAACT-5’5’ – ATGCCGGCTAAGACTGTAGCTTGA-3’

3’ – TAC GGC CGA TTC TGA CAT CGA ACT – 5’ 5’ - ATG CCG GCT AAG ACT GTA GCT TGA – 3’

mRNA – (codons)

tRNA – (anticodons)

Amino Acids =

AUG CCG GCU AAG ACU GUA GCU UGA

UAC GGC CGA UUC UGA CAU CGA ACU

Met – Pro – Ala – Lys – Thr – Val – Ala

Mutations: Changes in the DNA Sequence

Type of Mutation Typical Effect of Mutation

A. Chromosomal rearrangement (aneuploidy, trisomy, polyploidy)

A. Usually lethal due to high number of genes (proteins) involved

B. Changes in DNA base pairs (Point Mutations)

B. Effect Varies1. Silent – no effect due to the

redundancy of the triplet code2. Missense – Little effect resulting

with a functional protein3. Nonsense – Protein nonfunctional

1. Base- Pair Substitutions Replacing one base pair with another

C. Point Mutations Types

2. Deletions – removal of one base pair

3. Insertions – addition of one base pair

Usually silent due to redundancy and

“wobble” of code. Effect depends on:

1) Amino Acid type changed

2) Change in start or stop codons

Cause frame shift mutations forming nonsense proteins

THE END

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Ribosome Anatomy

Small Subunit

Large Subunit

mRNA

Growing Protein Chain

EP

A

Computer Model From X-Ray

Crystallography Data

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Difference in electronegativity of this amino acid causes hemoglobin to change shape after it gives up it’s O2

A Point Mutation with Bad Effects - Sickle Cell Anemia

No Change due to redundancy of the code or “wobble”

Different Amino Acid

Effect depends on the amino acid type

1. Hydrophobic or hydrophlic

2. Positive or negative

3. Acidic or basic

A premature stop codon will prevent from forming the complete protein

THEREDDOGANDCAT

THE RED DOG AND CAT

Reading frame

Deletion frameshift

THE EDD OGA NDC AT

R

Insertion frameshift

THE RRE DDO GAN DCA T

R

Genetic Code Redundancy

1. There are multiple codes for the same amino acids

2. The most variation is found in the 3rd base.

4. Some tRNA use the base Inosine(I) as their 3rd base that can bond to any base

“Wobble”

3. Some base pairs will bond to its “wrong” pair in the 3rd base

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