12From DNA to Protein: Genotype to Phenotype

Biochemical Biosynthesis Pathways Lead to Understanding of Gene-Enzyme Relationship

Biosynthesis of Arginine

Beadle & Tatum undertook the identification of mutations that blocked the synthesis of several vitamins & amino acids

Ornithinetranscarbamylase

Argininosuccinate synthetase

Argininosuccinate lyase

CitrullineOrnithineN-acetylornithine argininosuccinate arginine

aspartatecarbamyl phosphate

Acetylornithinase

Mutagenesis Screen Identifies Link Between Genetic Element and Enzyme

Figure 14.24 Raven & Johnson, Biology 5th Ed

Mutagen

Individual Mutants Blocked at Distinct Enzymatic Steps in Arg Biosynthesis Pathway

What is a Gene

• A gene is a contiguous region of DNA that is transcribed

• The transcript (that which is transcribed) is an RNA molecule

• There are 3 types of genes & 3 types of RNA transcribed rRNA encoding genes rRNA (class I) protein encoding genes mRNA (class II) tRNA encoding genes tRNA (class III)

• In eukaryotic cells, each class of RNA is transcribed by a different RNA polymerase

Decoding The Coding Problem

• 1959-60 – F Crick, S Brenner, F Jacob, M Meselson, J Monod

Messenger Hypothesis RNA serves as intermediate btwn DNA & protein

synthesis Ribosomes associated with protein synthesis Heterogeneous RNA (hnRNA) found w/ & w/o ribosomes Is hnRNA or rRNA the messenger? Brenner, Jacob & Meselson did a 1 week experiment that

proved hnRNA was the message – renamed mRNA

DNA, RNA, and the Flow of Information• F. Crick coined phrase central dogma

DNA codes for RNA. RNA codes for protein.

• How is expression of gene controlled?

• How does information get from the nucleus to the cytoplasm?

• What is relationship btwn DNA nucleotide sequence & protein amino acid sequence?

Transcription Translation

Replication

Gene Expression: From Gene to Protein

Decoding The Coding Problem

• Crick proposed the Adaptor Hypothesis intermediate btwn mRNA & protein synthesis intermediate adapt (bind) to mRNA & “decode” the

message

• What is nature of genetic code? 1 to 1 2 to 1 3 to 1 4 to 1

• Nature of the adaptor? tRNA –necessary for translation tRNAs w/ amino acids attached

Aminoacylated tRNA (aa tRNA)

DNA, RNA, and the Flow of Information: The Central Dogma

• Gene expression The production of an ultimate gene product (RNA &/or

protein)

• The expression of a gene takes place in two steps:

Transcription – production of a single-stranded RNA copy of a segment of DNA

Translation – production of a protein from mRNA

• Gene product is therefore

rRNA or tRNA or protein (via mRNA)

Review of RNA

• RNA differs from DNA

single stranded

ribose

uracil

• RNA can exist in a double-stranded complex with either DNA, with itself, or with another RNA strand

• mRNA – encodes proteins

• rRNA – main constituent of ribosomes

• tRNA – transfer amino acids to ribosome and decode mRNA

• snRNA – splicing

• snoRNA – RNA modifications

• 7SL RNA – co-tranlational translocation for secretion

• siRNA – regulation of transcription & translation

Transcription: DNA-Directed RNA Synthesis

• Requirements:

A DNA template

ribonucleoside triphosphates (ATP, GTP, CTP, and UTP)

RNA polymerase

• Regulated process

transcription factors

DNA sequences recognized by RNA pol & txn facs

Gene Structure

• The DNA template Strand nomenclature

• For different genes in the same DNA molecule, the roles of the strands may be reversed

top, coding, sense5’

5’3’

3’

bottom, template, antisense

3’promoter coding region

5’5’3’coding strand

template strand

txn initiation site

Transcription: DNA-Directed RNA Synthesis

• Initiation RNA polymerase binds to the promoter region

Coding

Transcription: DNA-Directed RNA Synthesis

• Elongation RNA polymerase unwinds the DNA and synthesizes RNA Nucleotides added at 3’ end of growing RNA strand

5 3 Template and RNA transcript are antiparallel

Transcription: DNA-Directed RNA Synthesis

• Termination RNA polymerase reaches DNA sequences at end of gene

that cause it to stop and release the RNA and DNA

Transcription – Prok v Euk

• Prokaryotic transcription occurs ___________

• Eukaryotic transcription occurs ____________

• Prokaryotic cells have ___________ RNA polymerase

• Eukaryotic cells have ____________ RNA polymerases

The Genetic Code

• The genetic code relates nucleotide sequence of genes (DNA/mRNA) to the amino acid sequence of proteins

• What is the nucleotide-amino acid correspondence? A degenerate code Frames

• How many nucleotides correspond to an amino acid? Three A triplet code

The Genetic Codebreakers

• 1960-65 M Nirenberg, G Khorana, P Leder

• Identified which nucleotide sequences specified which amino acids

The Genetic Code

• A codon was determined to be 3 adjacent nucleotides

• Code is degenerate Multiple codons specify same amino acid Each of these codons is NOT recognized by a different

tRNA “wobble” in the base-pairing btwn tRNA anticodon w/

mRNA codon

The Genetic Code

Anticodons & Wobble

RNA-RNA bp rulesA-UG-C G-U

Modified basesInosine (I)

I-AI-UI-C

5’-codon-3’ / 3’-αcodon-5’

The Genetic Code

• How is the code read?

• 3letterwordsallruntogetherwhatspunctuation?

Translation: Reading Frames

AAGCUAGCAUGUGGAUGCAUGAUCGCUACAAUCGAGGAUC

a: AAG CUA GCA UGU GGA UGC AUG AUC GCU ACA AUC GAG GAU C

b: A AGC UAG CAU GUG GAU GCA UGA UCG CUA CAA UCG AGG AUC

c: AA GCU AGC AUG UGG AUG CAU GAU CGC UAC AAU CGA GGA UC

Lys Leu Ala Cys Gly Cys Met Ile Ala Thr Ile Glu Asp

Ser stop His Val Asp Ala stop Ser Leu Gln Ser Arg Ile

Ala Ser Met Trp Met His Asp Arg Tyr Asn Arg Gly

Putative Translation of cDNA Sequence

Translation - tRNA

• tRNA has three functions:

carries amino acid

base-pairs with mRNA

interacts with ribosomes

• tRNAs must read mRNA correctly to assure proper protein sequence

• tRNAs must carry the correct amino acids

Translation - tRNA

• Intramolecular base pairing defines 2 structure

Charging a tRNA Molecule

aminoacyl-tRNA synthetase

Amino acids attached to correct tRNAs by aminoacyl-tRNA synthetases

Phe-tRNAPhe

aa-tRNA

Translation – Ribosomes

• 2 subunits: Large & Small

• Eukaryotes Large – 60S – 28S, 5.8S & 5S rRNA + ~45 proteins Small – 40S – 18S rRNA + ~ 33 proteins Ribosome – 80S

• Prokaryotic Large – 50S – 23S & 5S rRNA + ~ 40 proteins Small – 30S – 16S rRNA + ~ 28 proteins Ribosome – 70S

Ribosomes

Electron Density Model of Ribosome & tRNAs

Translation - Ribosome

A site – aa-tRNA binding site P site - tRNA with peptide chain E site – exit site empty tRNA briefly sits after translocation

Translation Initiation

• Involves initiation factors to help ribosome & Met-tRNAi

Met bind

• Initiator tRNA enters P-site

Translation Elongation

• Elongation factors aa-tRNA binding Translocation

• Peptidyl transferase Ribozyme activity of

large subunit

aa-tRNA entry

Peptidyl transferase

Translocation Termination

Translation Elongation

Polysomes

Regulation of Translation

• Antibiotics defensive molecules produced by fungi & bacteria, against

other microbes

• Molecular modality synthesis of cell walls, inhibiting transcription, inhibiting

translation erythromycin streptomycin tetracycline

• Because of differences between prokaryotic and eukaryotic ribosomes, the human ribosomes are usually unaffected.

Posttranslational Events• Folding

• Glycosylation

• Phosphorylation

• Acylation

• Proteolytic processing

• Dimerization/multimerization

Postranslational Events

• Subcellular location of translation and ultimate protein localization & modification

Figure 12.15 A Signal Sequence Moves a Polypeptide into the ER (Part 1)

Figure 12.15 A Signal Sequence Moves a Polypeptide into the ER (Part 2)

AUGUGGCUCCCGAUUAA

Point Mutations

coding ATGTGGCTCCCGATTAAATGTGGCTCCTGATTAA

T C

AUGUGGCUCCUGAUUAA

AUGUGGCUCCCGAUUAA

Point Mutations

ATGTGGCTCCCGATTAAATGTGGCTCCCGTTTAAcoding

AUGUGGCUCCCGUUUAA

Point Mutations

ATGTGGCTCCCGATTAAATGTAGGCTCCCGATTAAcoding

Point Mutations

ATGTGGCTCCCGATTAAATGTGGACTCCCGATTAAcoding

Tautomeric Shifts Alter Base-Pairing Specificity

Keto-enol & amino-imino tautomerization

Mutations Arise from Chemical Changes in BasesDeamination

Alkylation