Transcript File

Protein synthesis decodes the information in
messenger RNA
Protein synthesis occurs in three phases:
1. Initiation – the translation machinery
locates the start codon in mRNA
2. Elongation – codons are read 5’  3’ as
the
protein is synthesized from the amino
end to the carboxyl end
3. Termination – special proteins hydrolyze
the polypeptide from the last tRNA when
Ribosomes have three tRNA-binding sites
that bridge the 30S and 50S subunits
The mRNA being translated is bound to 30S
Each tRNA molecule contacts both 30S and 50S
Two of the three tRNAs have anticodon-codon
interactions with the mRNA
A site = aminoacyl site
P site = peptidyl site
The E site (exit site) contains the third tRNA
tRNA acceptor stems are positioned in 50S
The start signal is AUG or GUG preceded by
several bases that pair with 16S rRNA
Nearly half the amino terminal aa residues in
proteins from E. coli are methionine,
suggesting that AUG is the start codon
Initiator regions contain a purine-rich sequence
called the Shine-Dalgarno sequence about
10 nucleotides 5’ of the initiator codon
The Shine-Dalgarno sequence interacts with a
complementary region on the 3’ end of 16S
RNA
Bacterial protein synthesis is initiated by
formylmethionyl (fmet) tRNA
The initiator tRNA (tRNAf) differs from tRNAm used
for internal methionine residues
The initiator fmet is removed from about half of
the proteins found in E. coli from the newly
synthesized protein
A single aminoacyl-tRNA synthetase links met
to both tRNA molecules, however met
attached to tRNAf is recognized by a specific
enzyme that formylates the met amino group
fMet-tRNAf is placed in the P site during
formation of the 70S initiation complex
Initiation factors IF1 and IF3 join the 30S subunit to
preventing 30S from prematurely binding 50S
IF2, a GTPase, binds GTP to change shape and
enable binding of IF2 to fMet-tRNAf.
The IF2-GTP- fMet-tRNAf complex binds mRNA
bound to 16S rRNA via theShine-Dalgarno
sequence to create the 30S initiation complex
Binding of 50S causes GTP hydrolysis, IF’s are
released and the 70S initiation complex is formed
Elongation factors deliver aminoacyl-tRNA to
the ribosome
The mRNA codon in the A site defines which
aminoacyl-tRNA will enter the site
Elongation Factor Tu (EF-Tu) delivers the correct
aminoacyl-tRNA to the A site when GTP is bound
EF-Tu serves two functions:
1. EF-Tu protects the ester linkage in aminoacyltRNA from hydrolysis
2. The GTP in EF-Tu is hydrolyzed to GDP only
when an appropriate complex between the
EF-Tu-aminoacyl-tRNA complex and the
ribosome has formed
EF-Tu is then reset to its GTP form by EF-Ts
which induces dissociation of GDP from EF-Tu
and replacement by GTP
Peptidyl transferase catalyzes peptide bond
formation
When both the A and P sites are occupied by
aminoacyl-tRNA, the formylmethionine linked to
initiator tRNA is transferred to the amino group in
the A site.
The peptidyl transferase center on the 23S subunit
of the 50S subunit catalyzes formation of the
peptide bond
Formation of a peptide bond is followed by GTPdriven translocation of tRNAs and mRNA
The translocation mechanism
Proteins are synthesized by the successive
addition of amino acids to the carboxyl terminus
Protein synthesis is terminated by release
factors that read stop codons
Stop codons (UAA, UGA or UAG) are read by
protein release factors
FR1 recognizes UAA or UAG and RF2 recognizes
UAA or UGA
RF3 is a GTPase that mediates interactions between
RF1 or RF2 and the ribosome
RF1 and RF2 mimic tRNAs and promote hydrolytic
attack on the ester linkage between tRNA and the
polypeptide
Prokaryotes and eukaryotes differ in the
initiation of protein synthesis
1. Eukaryotic ribosomes are larger, consisting of a
60S large subunit and a 40S small subunit. The
60S subunit contains three RNAs: 5S RNA, 28S
RNA and 5.8S RNA. The 40S subunit contains an
18S RNA.
2. The initiating amino acid in eukaryotes is
methionine rather than N-formylmethionine. A
special initiator tRNA is used called tRNAi
3. The initiating codon is always AUG with no
Shine-Dalgarno sequence
Prokaryotes and eukaryotes differ in the
initiation of protein synthesis
4. Eukaryotic mRNA is circular. eIF-4E protein
binds the 5’ 7-methylguanosine cap and the 3’
poly(A) tail through protein intermediates
Some antibiotics inhibit proteins synthesis
Streptomycin, a highly basic trisaccharide,
interferes with binding of formylmethionyl-tRNA
to ribosomes preventing initiation
Neomycin, kanamycin and gentamycin interfere with
interactions between tRNA and the 16S rRNA to
inhibit initiation
Choramphenicol inhibits peptidyl transferase
Erythromycin binds the 50S subunit and blocks
translocation
Ribosomes bound to the endoplasmic
reticulum manufacture secretory and
membrane proteins
Ribosomes attached to the endoplasmic reticulum
to form the Rough Endoplasmic Reticulum (RER)
Ribosomes on RER synthesize proteins destined to
exit the cell or become membrane proteins
exposed on the surface of the cell
Three classes: Secretory proteins; lysosomal
proteins; and proteins spanning the plasma
membrane
Protein synthesis begins on ribosomes that
are free in the cytoplasm
Secretory, lysosomal and membrane proteins begin
synthesis on a free ribosome but then arrest until
the ribosome binds the cytoplasmic surface of the
ER
Docking with the ER restarts protein synthesis with
the newly synthesized protein threaded into the
lumen of the ER
Signal sequences mark proteins for translocation
across the ER membrane
The translocation machinery consists of four
components:
1. The Signal Sequence – 9 to 12 hydrophobic
amino acid residues, sometimes with positively
charged amino acid residues, usually near the
amino terminus of the nascent peptide chain.
Some signal sequences are maintained in the
mature protein while others are cleaved by a
signal peptidase.
2. The Signal Recognition Particle (SRP) recognizes
and binds the signal peptide then directs the
peptide to the ER lumen. SRPs are GTPases.
The translocation machinery consists of four
components:
3. The SRP Receptor binds SRP at the surface of
the ER membrane. The SRP Receptor is a
GTPase.
4. The Translocon is the translocation machinery
that transports the nascent polypeptide across
the ER membrane.