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Making Proteins

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Page 1: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Making Proteins

Page 2: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

TRANSLATION

TRANSCRIPTION

Protein

mRNACytoplasm

mRNA

DNA

Nucleus Informationstorage

Informationcarrier

Active cellmachinery

Central Dogma of Genetics

Page 3: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

The Genetic Code

The nucleotide sequence of DNA is a code; DNA is an information-storage molecule without enzymatic capabilities(F. Crick).

The information in DNA is copied into RNA, which is used to make proteins (mRNA = messenger RNA).

Hypothesis: each of the 20 amino acids in proteins is specified by one or more 3 base codons (Gamow).

Page 4: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

There are 4 RNA bases (U, C, A, G) and they must specify 20 amino acids.

How many bases specifya single amino acid?

Since there are only 4 bases, a singlet codecould onlyspecify 4amino acids.

A doublet code couldspecify a maximum of4 x 4 or 16 amino acids.

A triplet code could specify amaximum of 4 x 4 x 4, or 64amino acids.

1 Base? 2 Bases? 3 Bases? 4 Bases?...

4 < 20: Not enough

16 < 20: Not enough 64 > 20: More than enough

1 2 3 4 1 2 3 4

5 6 7 8

9 10 11 12

13 14 15 16

1 2 3 4

5 6 7 8

9 10 11 12

13 14 15 etc...

U GC A U

G

C

A

U

G

C

A

U

G

C

A

U

G

C

A

U

G

C

A

U

G

C

A

U

G

C

A

U

C

A

U C A G

U C A G

U C A G

U C A G

U C A G

U C A G

U C A G

U C A

U

G

C

A

U

G

C

A

U

G

C

A

U

C

A

mRNAGC CA

CC

G AGA A AA AA A AA AU U U UU U U U UC CC CC CC CCC C CGG G

GG G

G

G

How does the genetic code work?

Page 5: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.4 The dictionary of the genetic code

Page 6: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

One gene-one polypeptide hypothesis:

A gene is a length of a DNA molecule that

contains the information to produce one polypeptide

chain

Page 7: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 1)

Page 8: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 2)

Page 9: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 3)

Page 10: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 4)

Page 11: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 5)

Page 12: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

5’

3’5’

3’

3’

5’

3’5’

DNARNA

PP

P

O

OH

OH

O

OH

OH 3’P P PP

5’

RN

A

OHO

OOH

OH

AU

A T CG

C

5’3’

O O O O

P P P PG

DN

A

Transcription produces an RNA molecule complementary to a DNA template

Template strand

Page 13: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

RNApolymerase DNA

RNA transcription is catalyzed by RNA polymerase

Page 14: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Protein Synthesis Begins with the Process ofGene Transcription

Steps of Transcription

• RNA polymerase binds to the promoter region of the DNA

• RNA polymerase unwinds the DNA.

• RNA polymerase reads DNA 3' to 5' and synthesizes complementary RNA 5' to 3'.

Page 15: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 1)

Page 16: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 2)

Page 17: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 3)

Page 18: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 4)

Page 19: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Close up of transcription

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Page 20: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

In eukaryotes: proteins called transcription factors bind to the promoter first, then RNA polymerase binds to start transcription

Page 21: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

After Transcription

Transcription in Prokaryotes

• The RNA produced is ready to be translated = mRNA

Transcription in Eukaryotes

• The RNA produced must be modified before translation: 1° transcript--> mRNA

• Eukaryotic mRNAs are processed in the nucleus by additionof a 5' cap and 3' poly A tail

• Eukaryotic genes have introns: non-coding regions thatmust be removed from the primary mRNA to make an intact uninterrupted message.

Page 22: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

RNA processing in Eukaryotes

Page 23: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Molecules called small nuclear ribonucleoproteins (snRNPs) combine to splice

introns from mRNA

Page 24: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.11 Correspondence between exons and protein domains

Page 25: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

After transcription, the next step is translation

Translation Converts the Nucleotide Sequence of mRNA into the Amino Acid Sequence of a Protein

Translation occurs on ribosomes either in the cytoplasm or on the endoplasmic reticulum

Page 26: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Active site(containsonly rRNA)

A site

P site

Large subunitSmall subunit

rRNAs = ribosomal RNA

Proteins

Structure of a ribosome

E site

Page 27: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Loops consistof unpairedbases

The adaptor molecule between mRNA and protein is tRNA (transfer RNA)

Stems are createdby hydrogen bondingbetween complementarybase pairs

Page 28: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.13b The structure of transfer RNA (tRNA)

Page 29: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA

Page 30: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Ser

ACC

3’

5’Binding site foramino acid

Binding site formRNA codon

Aminoacid

GA U

U CA

Serine anticodon

Serine codon

Early model of tRNA function

5’ 3’

mRNA

Page 31: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.15 The anatomy of a functioning ribosome

Page 32: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Translation Converts the Nucleotide Sequence of mRNA into the Amino Acid Sequence of a Protein

Translation occurs in three steps:

• Initiation: the ribosome 30S subunit binds mRNA and movesto the AUG codon, which is the translation start site.

• The initiator methionine tRNA binds to the AUG start codon.

• The ribosome 50S subunit assembles so that the initiator tRNA and the AUG codon are in the P site.

Page 33: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.17 The initiation of translation

Page 34: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Translation Converts the Nucleotide Sequence of mRNA into the Amino Acid Sequence of a Protein

Translation occurs in three steps:

• Elongation: amino acids are joined together and the ribosome moves to the next codon.

• New tRNAs enters the A site of the ribosome

• A peptide bond forms between the polypeptide on the tRNA inthe P site and the amino acid in the A site, which transfers the polypeptide to the A site tRNA.

• The ribosome moves along the mRNA in the 5' to 3' direction.

Page 35: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.18 The elongation cycle of translation

Page 36: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Translation Converts the Nucleotide Sequence of mRNA into the Amino Acid Sequence of a Protein

Translation occurs in three steps:

• Termination: when a stop codon on mRNA is encountered in the A site, the completed polypeptide is released, and the ribosome disengages.

• Release factors are required.

Page 37: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.19 The termination of translation

Page 38: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Post-translational events affect the structure, activity, and destination of the protein

Proteins must fold into their proper 3D structure.

Primary structure

Secondary structure

Tertiary structure

Quaternary structure

Page 39: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Virus protein coat

Host cell membrane

Virus RNA

1. Start of infection.Virus RNA enters hostcells.

2. Reverse transcriptase uses Virus RNA as template to produce virus DNA

4. End of infection.New generation ofvirus particles burstfrom host cell.

The Central Dogma: Information Flows from DNA to RNA to Proteins (F.Crick)

Viruses that have RNA genomes contradict the centraldogma, but all cells conform to it.

3. Virus DNA directsthe production of newvirus particles.

Page 40: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Mutation and DNA Repair Mechanisms

Mutations are created by chemicals, radiation, errors in meiosis and mistakes in DNA replication.

• Mutations can be deleterious, beneficial, or silent.

• Mutations in an individual are usually deleterious, may cause disease and death.

• Mutations in a population are a source of genetic diversity that allows evolution to occur.

Page 41: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

A A C T G G C

T T G A C C G

A A C T G G C

T T G A T C G

T T G A C C G

A A C T G G C

A A C T G G C

T T G A C C G

T T G A T C G

A A C T A G C

A A C T G G C

T T G A C C G

T T G A C C G

A A C T G G C

Parental DNA

DNA replication

First generation progeny

3'

5'

5'

3'

Second generation progeny

Wild type

MUTANT

Wild type

Wild type

DNA replication

Point mutations are a change in single base pair of DNA

A base-pair substitution:

Page 42: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Figure 17.24 Categories of Base-pair substitutions

Page 43: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

DNA sequence

Amino acid sequence

DNA sequence

Amino acid sequence

CACGTG

GTGCAC

GACCTG

TGAACT

GGACCT

CTCGAG

CTCGAG

CACGTG

GTGCAC

GACCTG

TGAACT

GGACCT

CTCGAG

ValineHistidine

LeucineThreonine

Proline Glutamic acid

Glutamic acid

CACGTG

ValineHistidine

LeucineThreonine

Proline ValineGlutamic acid

Normal

Mutant

Start of coding sequence

Normal red blood cells

Sickled red blood cells

DNA point mutations can lead to a different amino acid sequence.

Phenotype

Page 44: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Insertion or deletion of a single base-pair causes

frameshift mutations

Page 45: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

DNA strand with adjacent thymine bases

P

CH2

P

CH2

P

O

O N

N

O

H CH3

O

HN

O

H CH3

O

HN

Thymine

Thymine

UV light

P

CH2

P

CH2

P

O

ON

N

O

H CH3

O

HN

O

H CH3

O

HN

Thymine dimer

Kink

UV-induced thymine dimers caused DNA to kink

UV radiation can cause 2 thymines that are next to each other to bind to each other instead of the adenines in the other strand

Page 46: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Mutation and DNA Repair Mechanisms

DNA Repair Mechanisms

• DNA polymerase proofreads and corrects point mutations during replication.

• Other excision repair systems scan newly formed DNA and correct remaining mutations.

• Repair enzymes identify the correct template strand by its methyl groups.

• Defects in repair system enzymes are implicated in a variety of cancers.

Page 47: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

3'

5'

3'

5'

T G T C CA

T C G C

A C A G GG

OH 3'

T G T C C A T C G C

A C A G G

GT

5'

5'

OH 3'

OH

Mismatched bases.

Polymerase III can repair mismatches.

DNA polymerase proofreads DNA during replication

Page 48: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

1. Where a mismatch occurs, the correct base is located on the methylated strand: the incorrect base occurs on the unmethylated strand.

2. Enzymes detect mismatch and nick unmethylated strand.

3. DNA polymerase I excises nucleotides on unmethylated strand.

4. DNA polymerase I fills in gap in 5'    3' direction.

5. DNA ligase links new and old nucleotides.

METHYLATION-DIRECTED MISMATCHED BASE REPAIR

Mismatch

Repaired Mismatch

Page 49: Making Proteins. TRANSLATION TRANSCRIPTION Protein mRNA Cytoplasm mRNA DNA Nucleus Information storage Information carrier Active cell machinery Central

Some genetic diseases are associated with mutations in DNA repair mechanisms

Xeroderma pigmentosum is a defect in ultraviolet radiation induced DNA repair mechanisms; characterized by severe sensitivity to all sources of UV radiation (especially sunlight). Symptoms include blistering or freckling, premature aging of skin,with increased cancers in these same areas, blindness resulting from eye lesions or surgery for skin lesions close to the eyes