Colinearity of Gene and Protein

DNA

RNA

protein

genotype

function

organismphenotype

DNA sequence

amino acidsequence

transcription

translation

“The linear sequence of nucleotides in a gene determines the linear sequence of amino acids in a protein.”

Mutant alleles of trpA gene differed in the position of the mutation at the DNA level, which corresponded to position of amino acid substitution in the gene product.

Colinearity of Gene and Protein

Colinearity of mutations and altered amino acids in subunit of tryptophan synthetase from E. coli

C. Yanofsky, 1967. Scientific American

Molecular Basis forRelationship between Genotype and Phenotype

DNA

RNA

protein

genotype

function

organismphenotype

DNA sequence

amino acidsequence

transcription

translation

Anticodon of a tRNA molecule recognizes and pairs with an mRNA codon.

tRNA contains modified bases: pseudouridine, methylguanosine, dimethylguanosine, methylinosine, dihydrouridine.

tRNA

Genetic Code

Aminoacyl-tRNA Synthetase Attaches Amino Acid to tRNA

Aminoacyl-tRNA synthetase catalyzes the formation of “charged” tRNA.

There is an aminoacyl-tRNA synthetase for each amino acid.

The carboxyl end of an amino acid is attached to the 3’ end of the tRNA.

Wobble Position

Some tRNA molecules can recognize and pair with more than one specific codon.

Base-pairing between the 3’ base of a codon and 5’ base of an anticodon is not always exact.

Molecular Basis forRelationship between Genotype and Phenotype

DNA

RNA

protein

genotype

function

organismphenotype

DNA sequence

amino acidsequence

transcription

translation

Protein Synthesis: Brief Summary

3 Stages

• Initiation• Elongation• Termination

Catalytic Proteins

• Initiation Factors• Elongation Factors• Termination Factors

Hydrolysis of GTP provides energy to drive some reactions.

mRNA, rRNA, and tRNA are involved.

Protein Synthesis: Initiation in Prokaryotes

Shine-Dalgarno sequence pairs with 16S rRNA of 30S subunit.

IF3 keeps 30S subunit dissociated from 50S subunit.

Formyl group is added to methionine when associated with the initiator tRNA.

IF1 and IF2 allows only initiator tRNA to enter P site.

Initiation factors are released when two ribosomal subunits associate.

Protein Synthesis:Initiation in Eukaryotes

eIF4A, eIF4B, and eIF4G associates with 5’ end, then with 40S subunit and initiator tRNA.

mRNA is unwound by movement of this complex in 5’ -> 3’ direction.

60S subunit associates with initiation complex when start codon is recognized.

Initiation factors are released when the two ribosomal subunits associate.

Important Features of Ribosome

A - aminoacyl site P - peptidyl site E - exit site

EF-Tu associates with aminoacyl-tRNA to form a ternary complex.

Protein Synthesis: Elongation

Amino acid in P site is transferred to amino acid in A site.

Translocation requires GTP and EF-G. EF-G enters A site, shifting tRNAs. When EF-G leaves, A site is open for a new ternary complex. A new ternary complex associates with A site, and deacylated tRNA leaves from E site.

Correct match of ternary complex with codon in A site (decoding center) changes conformation of ribosome.

EF-Tu leaves ternary complex, and peptide bond is formed between amino acids as amino acids are positioned together in peptidyltransferase center.

Protein Synthesis: Termination

tRNA molecules do not recognize stop codons.

Termination codons are recognized by release factors. (RF1, RF2, RF3 in bacteria)

UAA and UAG are recognized by RF1.

UAA and UGA are recognized by RF2.

RF3 assists in release activity.

Release factors bind to a stop codon in the A site by association between codon and tripeptide of RF.

Polypeptide is released from P site when RF fits into A site.

Release of polypeptide is followed by dissociation of ribosomal subunits.

Molecular Basis forRelationship between Genotype and Phenotype

DNA

RNA

protein

genotype

function

organismphenotype

DNA sequence

amino acidsequence

transcription

translation

Molecular Basis forRelationship between Genotype and Phenotype

DNA

RNA

protein

genotype

function

organismphenotype

DNA sequence

amino acidsequence

transcription

translation

All Protein Interactions in an Organism Compose the Interactome

Proteome:

Complete set of proteins produced by genetic material of an organism.

Interactome:

Complete set of protein interactions in an organism.

Alternative Splicing Produces Related but Distinct Protein Isoforms

Posttranslational Events

Protein Folding:

Translational product (polypeptide) achieves appropriate folding by aid of chaperone proteins.

Modification of Amino Acids:

* Phosphorylation/dephosphorylation

* Ubiquitination

Protein Targeting:

Directing proteins to specific locations (for example, nucleus, mitochondria, or cell membrane) is accomplished by tagging of proteins (signal sequence for secreted proteins, nuclear localization sequences for nuclear proteins).

Posttranslational Events

Protein Folding:

Translational product (polypeptide) achieves appropriate folding by aid of chaperone proteins.

Modification of Amino Acids:

* Phosphorylation/dephosphorylation

* Ubiquitination

Protein Targeting:

Directing proteins to specific locations (for example, nucleus, mitochondria, or cell membrane) is accomplished by tagging of proteins (signal sequence for secreted proteins, nuclear localization sequences for nuclear proteins).

Phosphorylation and Dephosphorylation of Proteins

Kinases add phosphate groups to hydroxyl groups of amino acids such as serine and threonine.

Phosphatases remove phosphate groups.

Ubiquitinization Targets a Protein for Degradation

Short-lived proteins are ubiquitinated:

• cell-cycle regulators

• damaged proteins

Posttranslational Events

Protein Folding:

Translational product (polypeptide) achieves appropriate folding by aid of chaperone proteins.

Modification of Amino Acids:

* Phosphorylation/dephosphorylation

* Ubiquitination

Protein Targeting:

Directing proteins to specific locations (for example, nucleus, mitochondria, or cell membrane) is accomplished by tagging of proteins (signal sequence for secreted proteins, nuclear localization sequences for nuclear proteins).

Signal Sequences Target Proteins for Secretion

Signal sequence at the amino-terminal end of membrane proteins or secretory proteins are recognized by factors and receptors that mediate transmembrane transport. Signal sequence is cleaved by signal peptidase.

Nuclear localization sequences (NLSs) are located in interior of proteins such as DNA and RNA polymerases. They are recognized by nuclear pore proteins for transport into nucleus.

Universality of Genetic Information Transfer

Genetic code is essentially identical for all organisms.

There are exceptions.

System AUA UGA

“universal” isoleucine terminationmammalian mitochondria methionine tryptophanyeast mitochondria isoleucine tryptophan

Comparison of Gene Expression

Prokaryotes

One type of RNA polymerase synthesizes all RNA molecules.

mRNA is translated during transcription.

Genes are not split. They are continguous segments of DNA.

mRNAs are often polycistronic.

Eukaryotes

Three different types of RNA polymerases synthesize different classes of RNA.

mRNA is processed before translation.

Genes are often split. They are not continguous segments of coding sequences.

mRNAs are mostly monocistronic.