Modification of Amino Acids

advertisement
Colinearity of Gene and Protein
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Colinearity of Gene and Protein
“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 mutations and altered amino acids in
a subunit of tryptophan synthetase from E. coli
C. Yanofsky, 1967. Scientific American
Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
tRNA
Anticodon of a tRNA molecule recognizes and pairs with an mRNA codon.
tRNA contains modified bases: pseudouridine, methylguanosine, dimethylguanosine,
methylinosine, dihydrouridine.
Genetic Code
Aminoacyl-tRNA Synthetase Attaches Amino Acid to tRNA
Aminoacyl-tRNA
synthetase catalyzes the
formation of “charged”
tRNA.
There is an aminoacyltRNA 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 for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
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
Protein Synthesis:
Elongation
EF-Tu associates with
aminoacyl-tRNA to form
a ternary complex.
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.
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.
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 for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
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
“universal”
mammalian mitochondria
yeast mitochondria
AUA
isoleucine
methionine
isoleucine
UGA
termination
tryptophan
tryptophan
Comparison of Gene Expression
Prokaryotes
Eukaryotes
One type of RNA polymerase
synthesizes all RNA molecules.
Three different types of RNA
polymerases synthesize different
classes of RNA.
mRNA is translated during
transcription.
mRNA is processed before
translation.
Genes are not split. They are
continguous segments of DNA.
Genes are often split. They are not
continguous segments of coding
sequences.
mRNAs are often polycistronic.
mRNAs are mostly monocistronic.
Download