Transcript the amino acid

Mary K. Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell
Chapter Twelve
Protein Synthesis: Translation of
the Genetic Message
Paul D. Adams • University of Arkansas
Translating the Genetic Message
• Protein biosynthesis is a
complex process
requiring ribosomes,
mRNA, tRNA, and
protein factors
• Several steps are
involved
• Before being
incorporated into
growing protein chain,
a.a. must be activated
by tRNA and
aminoacyl-tRNA
synthetases
The Genetic Code
• Salient features of the genetic code
• triplet: a sequence of three bases (a codon) is
needed to specify one amino acid
• nonoverlapping: no bases are shared between
consecutive codons
• commaless: no intervening bases between codons
• degenerate: more than one triplet can code for the
same amino acid; Leu, Ser, and Arg, for example, are
each coded for by six triplets
• universal: the same in viruses, prokaryotes, and
eukaryotes; the only exceptions are some codons in
mitochondria
The Genetic Code (Cont’d)
• The ribosome moves
along the mRNA three
bases at a time rather
than one or two at a
time
• Theoretically possible
genetic codes are
shown in figure 12.2
The Genetic Code (Cont’d)
• All 64 codons have assigned meanings
•
•
•
•
61 code for amino acids
3 (UAA, UAG, and UGA) serve as termination signals
only Trp and Met have one codon each
the third base is irrelevant for Leu, Val, Ser, Pro, Thr,
Ala, Gly, and Arg
• the second base is important for the type of amino
acid; for example, if the second base is U, the amino
acids coded for are hydrophobic
• for the 15 amino acids coded for by 2, 3, or 4 triplets,
it is only the third letter of the codon that varies. Gly,
for example, is coded for by GGA, GGG, GGC, and
GGU
The Genetic Code (Cont’d)
The Genetic Code (Cont’d)
• Assignments of triplets in genetic code based on
several different experiments
• synthetic mRNA: if mRNA is polyU, polyPhe is
formed; if mRNA is poly --ACACACACACACACACACACA---, poly(Thr-His) is
formed
• binding assay: aminoacyl-tRNAs bind to ribosomes
in the presence of trinucleotides
• synthesize trinucleotides by chemical means
• carry out a binding assay for each type of
trinucleotide
• aminoacyl-tRNAs are tested for their ability to bind
in the presence of a given trinucleotide
The Filter-Binding Assay Helps Elucidate
the Genetic Code
Wobble Base Pairing
• Some tRNAs bond to one codon exclusively, but
many tRNAs can recognize more than one codon
because of variations in allowed patterns of
hydrogen bonding
• the variation is called “wobble”
• wobble is in the first base of the anticodon
Base Pairing Combination in the Wobble
Scheme
Wobble Base Pairing
Wobble Base Pairing Hypothesis
• The wobble hypothesis provides insight into some
aspects of the degeneracy of the code
• in many cases, the degenerate codons for a given
amino acid differ only in the third base; therefore
fewer different tRNAs are needed because a given
tRNA can base-pair with several codons
• the existence of wobble minimizes the damage that
can be caused by a misreading of the code; for
example, if the Leu codon CUU were misread CUC or
CUA or CUG during transcription of mRNA, the codon
would still be translated as Leu during protein
synthesis
Amino Acid Activation
• Amino acid activation
and formation of the
aminoacyl-tRNA take
place in two separate
steps
• Both catalyzed by
amionacyl-tRNA
synthetase
• Free energy of
hydrolysis of ATP
provides energy for
bond formation
Amino Acid Activation (Cont’d)
• This two-stage reaction allows selectivity at two
levels
• the amino acid: the aminoacyl-AMP remains bound
to the enzyme and binding of the correct amino acid is
verified by an editing site in the tRNA synthetase
• tRNA: there are specific binding sites on tRNAs that
are recognized by aminoacyl-tRNA synthetases.
tRNA Tertiary Structure
• There are several recognition sites for various amino
acids on the tRNA
Chain Initiation
• In all organisms, synthesis of polypeptide chain
starts at the N-terminal end, and grows from Nterminus to C-terminus
• Initiation requires:
•
•
•
•
•
•
tRNAfmet
initiation codon (AUG) of mRNA
30S ribosomal subunit
50S ribosomal subunit
initiation factors IF-1, IF-2, and IF-3
GTP, Mg2+
• Forms the initiation complex
The Initiation Complex
Chain Initiation
• tRNAmet and tRNAfmet contain the triplet 3’-UAC-5’
• Triplet base pairs with 5’-AUG-3’ in mRNA
• 3’-UAC-5’ triplet on tRNAfmet recognizes the AUG
triplet (the start signal) when it occurs at the beginning
of the mRNA sequence that directs polypeptide
synthesis
• 3’-UAC-5’ triplet on tRNAmet recognizes the AUG
triplet when it is found in an internal position in the
mRNA sequence
• Start signal is preceded by a Shine-Dalgarno purinerich leader segment, 5’-GGAGGU-3’, which usually
lies about 10 nucleotides upstream of the AUG start
signal and acts as a ribosomal binding site
Chain Elongation
• Uses three binding sites for tRNA present on the
50S subunit of the 70S ribosome: P (peptidyl) site, A
(aminoacyl) site, E (exit) site.
• Requires
•
•
•
•
70S ribosome
codons of mRNA
aminoacyl-tRNAs
elongation factors EF-Tu (Elongation factor
temperature-unstable), EF-Ts (Elongation factor
temperature-stable), and EF-G (Elongation factorGTP)
• GTP, and Mg2+
Shine-Dalgarno Sequence Recognized by
E. Coli Ribosomes
Elongation Steps
• Step 1
• an aminoacyl-tRNA is bound to the A site
• the P site is already occupied
• 2nd amino acid bound to 70S initiation complex. Defined by the
mRNA
•
Step 2
• EF-Tu is released in a reaction requiring EF-Ts
• Step 3
• the peptide bond is formed, the P site is uncharged
• Step 4
•
•
•
•
the uncharged tRNA is released
the peptidyl-tRNA is translocated to the P site
EF-G and GTP are required
the next aminoacyl-tRNA occupies the empty A site
Chain Elongation
Chain Termination
• Chain termination requires
• stop codons (UAA, UAG, or UGA) of mRNA
• RF-1 (Release factor-1) which binds to UAA and
UAG or RF-2 (Release factor-2) which binds to UAA
and UGA
• RF-3 which does not bind to any termination codon,
but facilitates the binding of RF-1 and RF-2
• GTP which is bound to RF-3
• The entire complex dissociates setting free the
completed polypeptide, the release factors, tRNA,
mRNA, and the 30S and 50S ribosomal subunits
Chain Termination
Components of Protein Synthesis
Protein Synthesis
• In prokaryotes, translation begins very soon after
mRNA transcription
• It is possible to have several molecules of RNA
polymerase bound to a single DNA gene, each in a
different stage of transcription
• It is also possible to have several ribosomes bound to
a single mRNA, each in a different stage of translation
• Polysome: mRNA bound to several ribosomes
• Coupled translation: the process in which a
prokaryotic gene is being simultaneously transcribed
and translated
Simultaneous Protein Synthesis on
Polysomes
• A single mRNA molecule is translated by several
ribosomes simultaneously
• Each ribosome produces a copy of the polypeptide
chain specified by the mRNA
• When protein has been completed, the ribosome
dissociates into subunits that are used again in
protein synthesis
Simultaneous Protein Synthesis on
Polysomes (Cont’d)
Eukaryotic Translation
• Chain Initiation:
• the most different from process in prokaryotes
• 13 more initiation factors are given the designation eIF
(eukaryotic initiation factor) (Table 12.4)
Eukaryotic Translation (Cont’d)
• Chain elongation
• uses the same mechanism of peptidyl transferase and
ribosome translocation as prokaryotes
• there is no E site on eukaryotic ribosomes, only A and
P sites
• there are two elongation factors, eEF-1 and eEF-2
• eEF2 is the counterpart to EF-G, which causes
translocation
• Chain termination
• stop codons are the same: UAG, UAA, and UGA
• only one release factor that binds to all three stop
codons
Posttranslational Modification
• Newly synthesized polypeptides are frequently modified
before they reach their final form where they exhibit biological
activity
• N-formylmethionine in prokaryotes is cleaved
• specific bonds in precursors are cleaved, as for example,
preproinsulin to proinsulin to insulin
• leader sequences are removed by specific proteases of the
endoplasmic reticulum; the Golgi apparatus then directs the
finished protein to its final destination
• factors such as heme groups may be attached
• disulfide bonds may be formed
• amino acids may be modified, as for example, conversion of
proline to hydroxyproline
• other covalent modifications; e.g., addition of carbohydrates
Examples of Posttranslational Modification
Protein Degradation
• Proteins are in a dynamic state and are often turned
over
• Degradative pathways are restricted to
• subcellular organelles such as lysosomes
• macromolecular structures called proteosomes
• In eukaryotes, ubiquitinylation (becoming bonded
to ubiquitin) targets a protein for destruction
• protein must have an N-terminus
• those with an N-terminus of Met, Ser, Ala, Thr, Val,
Gly, and Cys are resistant
• those with an N-terminus of Arg, Lys, His, Phe, Tyr,
Trp, Leu, Asn, Gln, Asp, Glu have short half-lives
Ubiquitin-Proteosome Degradation
Acidic N-termini Induced Protein
Degradation