Chap 12 Translation

8/17/2019 Chap 12 Translation

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Paul D. Adams • University of Arkansas

Mary K. Campbell

Shawn O. Farrellinternational.cengage.com/

Chapter Twelve

Protein Synthesis: Translation of

the Genetic Message


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Translating the Genetic Message

• Protein biosynthesis is acomplex processrequiring ribosomes,

mRNA, tRNA, andprotein factors

• Several steps are

involved

• Before beingincorporated intogrowing protein chain,a.a. must be activatedby tRNA andaminoacyl-tRNA

synthetases


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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


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The Genetic Code (Cont’d)

• The ribosome moves

along the mRNA three

bases at a time ratherthan one or two at a

time

• Theoretically possible

genetic codes are

shown in figure 12.2


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• 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 aminoacid; for example, if the second base is U, the aminoacids 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, andGGU


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• Assignments of triplets in genetic code based on

several different experiments

• synthetic mRNA: if mRNA is polyU, polyPhe isformed; 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 oftrinucleotide

• aminoacyl-tRNAs are tested for their ability to bind

in the presence of a given trinucleotide


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The Filter-Binding Assay Helps Elucidate

the Genetic Code


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Wobble Base Pairing

• Some tRNAs bond to one

codon exclusively, but

many tRNAs can recognizemore 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


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Base Pairing Combination in the Wobble

Scheme


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Wobble Base Pairing Alternatives


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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 givenamino 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 codonwould still be translated as Leu during protein

synthesis


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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


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Amino Acid Activation (Cont’d)

• This two-stage reaction allows selectivity at two

levels

• the amino acid: the aminoacyl-AMP remains boundto 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.


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tRNA Tertiary Structure

• There are several recognition sites for various aminoacids on the tRNA


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Chain Initiation

• In all organisms, synthesis of polypeptide chain

starts at the N-terminal end, and grows from N-

terminus 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


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The Initiation Complex


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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 beginningof the mRNA sequence that directs polypeptidesynthesis

• 3’-UAC-5’ triplet on tRNAmet

recognizes the AUGtriplet when it is found in an internal position in themRNA sequence

• Start signal is preceded by a Shine-Dalgarno purine-

rich leader segment, 5’-GGAGGU-3’, which usuallylies about 10 nucleotides upstream of the AUG startsignal and acts as a ribosomal binding site


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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 factortemperature-stable), and EF-G (Elongation factor-

GTP)

• GTP, and Mg2+


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Shine-Dalgarno Sequence Recognized by

E. Coli Ribosomes


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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


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Chain Elongation


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Chain Termination

• Chain termination requires

• stop codons (UAA, UAG, or UGA) of mRNA

• RF-1 (Release factor-1) which binds to UAA andUAG 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


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Chain Termination


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Components of Protein Synthesis


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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 transcribedand translated


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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


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Simultaneous Protein Synthesis on

Polysomes (Cont’d)


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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)


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Eukaryotic Translation (Cont’d)

• Chain elongation

• uses the same mechanism of peptidyl transferase andribosome translocation as prokaryotes

• there is no E site on eukaryotic ribosomes, only A andP sites

• there are two elongation factors, eEF-1 and eEF-2

• eEF2 is the counterpart to EF-G, which causestranslocation

• Chain termination

• stop codons are the same: UAG, UAA, and UGA• only one release factor that binds to all three stopcodons


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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


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Examples of Posttranslational Modification


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Protein Degradation

• Proteins are in a dynamic state and are often turnedover

• 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


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Ubiquitin-Proteosome Degradation

Acidic N termini Induced Protein


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Acidic N-termini Induced Protein

Degradation

Chap 12 Translation

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