Unit 8

The coding ratio. Given that there are four

different bases, what is the minimum number of

nucleotides per amino acid required to specify all

20 amino acids ?

minimum = 3 nucleotides per amino acids = triplet code

Define "codon"

A sequence of three nucleotides in mRNA that specifies a particular amino acid or a stop signal during protein synthesis.

Is the code overlapping or non-overlapping? Use Figs. 27-3 and 27-4 (p. 1007) to

distinguish between each.

Non-overlapping code

• Each nucleotide is part of only one codon.

• Codons are read sequentially in groups of three.

Overlapping code (not how real genetic code works)

• A nucleotide would be shared between multiple codons.

Example:
AUA
UAC
ACG

• A single base change would affect several amino acids.

define a reading frame

The way mRNA nucleotides are grouped into codons (sets of three bases) during translation.

• mRNA is read 5′ → 3′.

• There are three possible reading frames depending on where translation begins.

• Each frame produces different codon groupings and therefore different amino acid sequences.

• The correct reading frame is set by the start codon (AUG).

Are individual codewords delimited, as for example words in a sentence are

delimited by spaces

No. Individual codewords are not delimited.

• The genetic code is comma-less (no spaces or punctuation between codons).

• mRNA is read continuously in groups of three nucleotides.

• Codons are determined only by the reading frame set by the start codon (AUG).

What establishes the reading frame in RNA (pp. 1007 - 1008)?

• The start codon (AUG) on mRNA sets the reading frame.

• AUG codes for methionine (Met).

• The ribosome begins translation at AUG and then reads nucleotides in triplets (codons) from that point onward.

• This determines how all downstream codons are grouped.

Given a sequence of RNA, an indication of

the correct reading frame, and the genetic

code (Fig. 27-7, p. 1009), deduce the

relevant polypeptide sequence encoded in

that reading frame

• an RNA sequence

• the correct reading frame

• the genetic code table

Do this:

1. Read the RNA 5′ → 3′

2. Separate the sequence into codons (triplets) using the given reading frame

3. Translate each codon with the genetic code table

4. Start at AUG if the question is asking for the translated protein

5. Stop when you reach a stop codon: UAA, UAG, or UGA

Important notes

• AUG = Met and usually starts translation

• Stop codons do not code for an amino acid

• A different reading frame gives a different amino acid sequence

What are the expected consequences of a one or two base addition or deletion within the

above sequence

A one- or two-base insertion or deletion causes a frameshift mutation.

Consequences:

• The reading frame shifts, changing how codons are grouped.

• All codons downstream of the mutation change.

• This usually produces a completely different amino acid sequence.

• Often results in a premature stop codon, producing a short, nonfunctional protein.

Why are such additions or deletions (when they occur in the DNA) called frameshift

mutations?

They are called frameshift mutations because the addition or deletion of nucleotides shifts the reading frame of the codons.

Explanation:

• Codons are read in groups of three nucleotides.

• If 1 or 2 bases are added or deleted, the grouping of all downstream nucleotides changes.

• This shifts the reading frame, so different codons are read, producing a different amino acid sequence and often a premature stop codon.

initiation codon , temrination codon , open reading frame

1) Initiation codon

• The start codon (AUG) that signals the beginning of translation.

• Codes for methionine (Met) and establishes the reading frame.

2) Termination codon

• A stop codon that signals the end of translation.

• UAA, UAG, UGA.

• These do not code for an amino acid.

3) Open reading frame (ORF)

• A continuous sequence of codons beginning with a start codon (AUG) and ending with a stop codon.

• Represents the region of mRNA that can be translated into a polypeptide.

What does degeneracy refer to in the context of coding (Table 27-3 [p. 1009], and

p. 1009)?

• Degeneracy means that more than one codon can specify the same amino acid.

• Although there are 64 possible codons, they encode only 20 amino acids, so several codons correspond to the same amino acid.

• These codons are called synonymous codons.

Example:

• Leucine (Leu) → 6 codons

• Arginine (Arg) → 6 codons

• Glycine (Gly) → 4 codons

Key point for exams:

• Degeneracy usually occurs because the third base of the codon can vary (wobble position) without changing the amino acid.

Use Fig. 27-8 (p. 1012) to describe how aminoacyl tRNAs interact with mRNA.

Point out the anticodon on the tRNA and the codon on the mRNA.

How aminoacyl-tRNAs interact with mRNA:

• An aminoacyl-tRNA carries a specific amino acid attached to its 3′ end.

• The anticodon on the tRNA (a sequence of 3 nucleotides) base-pairs with a complementary codon on the mRNA.

• The codon is the 3-nucleotide sequence on the mRNA read 5′ → 3′.

• The anticodon on tRNA binds antiparallel and complementary to the codon.

Example (from figure):

• mRNA codon: AUC (5′ → 3′)

• tRNA anticodon: UAG (3′ → 5′)

Result:

• Correct tRNA binds the codon.

• The amino acid carried by that tRNA is added to the growing polypeptide chain.

Is the genetic code almost universal

Yes, the genetic code is almost universal.

• Nearly all organisms use the same codons to specify the same amino acids.

• This means a gene from one organism can often be correctly translated in another organism.

Exceptions:

• Some mitochondria

• Certain protozoa and microorganisms

In these cases, a few codons are assigned different amino acids or stop signals.

The genetic code has been conserved through evolutionary time because it is resistant to the

deleterious effects of mutations. Discuss, and give an example of, how the genetic code protects

against missense mutations at both the 5' and 3' nucleotide positions in the codon (pp. 1012 - 1013).

The genetic code protects against missense mutations because similar codons often encode the same or chemically similar amino acids.

3′ position of the codon (third base)

• The third base is highly degenerate (wobble position).

• A mutation here often does not change the amino acid → silent mutation.

Example:

• UUU → UUC

• Both code for Phe (phenylalanine).

• A mutation at the 3′ base does not change the amino acid.

5′ position of the codon (first base)

• A change at the first base may change the amino acid, but often to one with similar chemical properties, reducing functional damage.

Example:

• GAU → Asp (aspartate)

• AAU → Asn (asparagine)

• Both are polar amino acids with similar properties, so the protein function may be less affected.

Key idea

• Degeneracy and chemical similarity of encoded amino acids help minimize harmful effects of mutations, which is why the genetic code has been conserved through evolution.

Use Table 27-4 (p. 1012) to discuss the meaning of "wobble" (pp. 1010 - 1012)? Do not confuse

degeneracy with wobble. Degeneracy is a feature of the genetic code (some amino acids are encoded

for by more than one codon), while wobble is a feature of a tRNA (some tRNAs can bind to more than

one codon).

Use Table 27-4 (p. 1012) to discuss the meaning of "wobble" (pp. 1010 - 1012)? Do not confuse

degeneracy with wobble. Degeneracy is a feature of the genetic code (some amino acids are encoded

for by more than one codon), while wobble is a feature of a tRNA (some tRNAs can bind to more than

one codon).

• Wobble refers to flexible base pairing between the 3′ base of the mRNA codon and the 5′ base of the tRNA anticodon.

• Because of this flexibility, one tRNA can recognize more than one codon.

Key points

• The first two bases of the codon pair normally (Watson–Crick pairing).

• The third base of the codon can pair less strictly, allowing alternative base pairing.

• This reduces the number of different tRNAs needed.

• Degeneracy: multiple codons encode the same amino acid (property of the genetic code).

• Wobble: a single tRNA can bind multiple codons (property of tRNA).

Use Fig. 27-10 (p. 1014) to show how guide RNAs can be used as templates to direct the

addition or removal of specific nucleotides from an mRNA transcript.

• Guide RNAs (gRNAs) base-pair with a pre-mRNA transcript.

• The gRNA acts as a template showing where nucleotides must be inserted or removed.

Process

1. gRNA binds to the mRNA by complementary base pairing.

2. Mismatches between gRNA and mRNA identify editing sites.

3. Enzymes then:

   • Insert nucleotides (commonly U residues) or

   • Delete nucleotides from the mRNA.

4. The edited mRNA now matches the guide RNA template.

Result

• The codon sequence changes, which can change the amino acid sequence of the protein produced.

Use Fig. 27-11 (p. 1014) to show how deaminases can alter specific nucleotides in an mRNA

transcript. Use Fig. 27-12 (p. 1015) to show how RNA editing by this mechanism is used to

regulate the production of two related but different products from a single transcript in a tissue-

specific manner.

1. How deaminases alter nucleotides in mRNA

Deaminase enzymes chemically modify bases by removing an amino group, changing the nucleotide identity.

Examples:

• Adenosine → Inosine (A → I)

  • Inosine is read by the ribosome as G.

• Cytidine → Uridine (C → U)

This changes the codon sequence, which can change the amino acid specified during translation.

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2. Example: tissue-specific RNA editing (ApoB protein)

RNA editing can produce different proteins from the same mRNA transcript.

Human liver:

• mRNA codon = CAA (Gln)

• Translation continues → produces ApoB-100 (full-length protein).

Human intestine:

• C → U editing converts CAA → UAA

• UAA is a stop codon

• Translation stops early → produces ApoB-48 (shorter protein).

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✅ Key idea for exam:
RNA editing by deaminases can change codons (including creating stop codons), allowing one gene to produce different proteins in different tissues.

activation of amino acids, initiation of protein synthesis, elongation , termination and ribosome recylcing , folding and post translational processing.

a. Activation of amino acids

• Each amino acid is attached to its specific tRNA by aminoacyl-tRNA synthetase.

• Uses ATP to form an aminoacyl-tRNA (charged tRNA).

• This prepares the amino acid for incorporation into the growing polypeptide.

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b. Initiation of protein synthesis

• Small ribosomal subunit binds mRNA near the start codon (AUG).

• Initiator tRNA carrying Met binds the start codon via its anticodon.

• Large ribosomal subunit joins, forming the complete ribosome.

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c. Elongation

• Aminoacyl-tRNA enters the A site of the ribosome matching the codon.

• Peptide bond forms between the growing chain and the new amino acid.

• Ribosome translocates along mRNA (5′ → 3′) and the cycle repeats.

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d. Termination and ribosome recycling

• A stop codon (UAA, UAG, UGA) enters the A site.

• Release factors bind, causing the polypeptide to be released.

• Ribosomal subunits, mRNA, and tRNA dissociate and are recycled.

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e. Folding and post-translational processing

• Newly made polypeptide folds into its functional 3-D structure (often with chaperone proteins).

• May undergo post-translational modifications such as:

  • Cleavage

  • Phosphorylation

  • Glycosylation

  • Disulfide bond formation.

Use Fig. 27-14 (pp. 1017) and Fig. 27-16 (p.

1018) to discuss the molecular composition and

three-dimensional conformation of the ribosome.

Bacterial ribosome (70S):

• Large subunit (50S):

  • rRNAs: 23S rRNA, 5S rRNA

  • ~36 proteins

• Small subunit (30S):

  • rRNA: 16S rRNA

  • ~21 proteins

Eukaryotic ribosome (80S):

• Large subunit (60S):

  • rRNAs: 28S rRNA, 5.8S rRNA, 5S rRNA

  • ~46 proteins

• Small subunit (40S):

  • rRNA: 18S rRNA

  • ~33 proteins

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Three-dimensional conformation of the ribosome

• The ribosome has two subunits that fit together to form a cleft where mRNA binds.

• tRNAs bind in three sites:

  • A site (aminoacyl site) – incoming aminoacyl-tRNA

  • P site (peptidyl site) – tRNA holding the growing polypeptide

  • E site (exit site) – tRNA leaving the ribosome

• The large subunit contains the peptidyl transferase center that forms peptide bonds.

• The small subunit binds and decodes the mRNA.

Use Fig. 27-15 (p. 1018) to illustrate that RNA (in addition to proteins) contributes

to this three-dimensional structure of the ribosome.

RNA contributes directly to the three-dimensional structure of the ribosome.

• rRNA forms the structural core of the ribosome, creating the main framework of the large and small subunits.

• Ribosomal proteins are mostly located on the outer surface, stabilizing the rRNA structure rather than forming the core.

• The complex folding of rRNA into helices and loops generates the ribosome’s 3-D architecture.

• rRNA also forms the peptidyl transferase center, which catalyzes peptide bond formation.

Key idea:
The ribosome is a ribozyme, meaning rRNA (not protein) provides both much of the structure and the catalytic activity of the ribosome.

Discuss the ribosome in the context of the RNA world hypothesis

Ribosome and the RNA world hypothesis

• The RNA world hypothesis proposes that early life relied primarily on RNA for both genetic information storage and catalytic activity before DNA and proteins evolved.

Evidence from the ribosome:

• The core structure of the ribosome is made mainly of rRNA, not protein.

• The peptidyl transferase center (where peptide bonds form) is catalyzed by rRNA, making the ribosome a ribozyme.

• Ribosomal proteins mainly stabilize the rRNA structure and are located on the outer surface.

Implication:

• Because the ribosome’s essential catalytic activity is carried out by RNA, it is considered a molecular relic of the RNA world, supporting the idea that RNA-based catalysts existed before protein enzymes evolved.

Why are tRNAs referred to as "adaptors"

tRNAs are called “adaptors” because they link the genetic code in mRNA to the correct amino acid during protein synthesis.

• Each tRNA has an anticodon that base-pairs with a specific codon on mRNA.

• The other end of the tRNA carries the corresponding amino acid.

• This allows the codon sequence in mRNA to be translated into the amino acid sequence of a protein.

the anticodon arm (or loop)

b. the TC arm

c. the dihydrouracil arm (D arm)

d. the 3' (CCA) and 5' ends of the tRNA molecule

e. the position at which the amino acid is attached

f. regions that are hydrogen bonded

a. Anticodon arm (loop)

• The bottom loop of the tRNA.

• Contains the anticodon (3 bases) that base-pairs with the codon on mRNA during translation.

b. TΨC arm

• The right arm of the tRNA containing the sequence T–Ψ–C (ribothymidine–pseudouridine–cytidine).

• Important for interaction with the ribosome.

c. D arm (dihydrouracil arm)

• The left arm of the tRNA containing dihydrouridine residues (D).

• Helps with recognition by aminoacyl-tRNA synthetase.

d. 3′ (CCA) and 5′ ends of the tRNA

• 3′ end: contains the CCA sequence, located at the top of the molecule.

• 5′ end: opposite side of the acceptor stem.

e. Position where the amino acid is attached

• The amino acid attaches to the 3′ hydroxyl of the terminal A in the CCA sequence at the 3′ end (acceptor stem).

f. Regions that are hydrogen bonded

• The stem regions of the cloverleaf structure (acceptor stem, D stem, anticodon stem, and TΨC stem).

• These regions form double-stranded sections stabilized by hydrogen bonding between complementary bases.

Amino acid activation (pp. 1020 - 1021).

Using structures, describe in two steps how

the activation of an amino acid for protein

synthesis takes place (Fig. 27-19, p. 1021).

Show the structures of the reactants,

intermediates, and the products

Step 1: Formation of aminoacyl-AMP (activation)

Reaction:

Amino acid+ATP→Aminoacyl-AMP+PP

• The carboxyl group of the amino acid forms an anhydride bond with AMP.

• Pyrophosphate (PPᵢ) is released.

Step 2: Transfer of the amino acid to tRNA

Reaction:Aminoacyl-AMP+tRNA→Aminoacyl-tRNA+AMP

The amino acid is transferred to the 3′ hydroxyl group of the terminal adenosine of the CCA end of tRNA.

Final products

• Aminoacyl-tRNA (charged tRNA)

• AMP

• PPᵢ

Name the class of enzyme that loads amino acids

onto tRNAs.

b. Name the linkage that is broken in ATP (refer to Fig.

1-14, p. 12)?

c. Name the linkage that is formed in the activated

intermediate which is then broken in the final stage

of the reaction (refer to Fig. 1-14, p. 12)?

d. Name the linkage that is ultimately formed between the

amino acid and the tRNA (refer to Fig. 1-14, p. 12)?

e. What is the other product of this series of reactions?

What happens to this product (p. 1020)?

a. Class of enzyme that loads amino acids onto tRNAs

• Aminoacyl-tRNA synthetases.

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b. Linkage broken in ATP

• The phosphoanhydride bond between the α and β phosphates of ATP is broken when ATP → AMP + PPᵢ.

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c. Linkage formed in the activated intermediate (then broken later)

• A mixed anhydride bond between the amino acid carboxyl group and AMP in aminoacyl-AMP (aminoacyl adenylate).

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d. Linkage ultimately formed between the amino acid and the tRNA

• An ester linkage between the carboxyl group of the amino acid and the 3′-OH of the terminal adenosine (CCA) of tRNA.

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e. Other product of the reaction and what happens to it

• Pyrophosphate (PPᵢ) is produced.

• It is rapidly hydrolyzed to 2 inorganic phosphates (2 Pᵢ) by pyrophosphatase, which drives the reaction forward (makes it essentially irreversible).

Write a balanced word equation for the sum of the activation and transfer steps, including hydrolysis of PPi (p. 1020).

g. There is a cost of two high energy anhydride bonds

in the synthesis of an aminoacyl-tRNA? Account

for this cost (pp. 1020).

h. Name two other synthetic processes you have

studied in which pyrophosphate is generated.

What is the significance of the fact that all cells

possess pyrophosphatases that catalyze the

hydrolysis reaction, PPi + H2O 2Pi?

f. Balanced word equation (including PPᵢ hydrolysis)

Amino acid + tRNA + ATP + H₂O → aminoacyl-tRNA + AMP + 2 Pi

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g. Why two high-energy bonds are used

• ATP is cleaved to AMP + PPᵢ, breaking one phosphoanhydride bond.

• The pyrophosphate (PPᵢ) is then hydrolyzed to 2 Pi, breaking another high-energy phosphoanhydride bond.

Thus the reaction consumes the equivalent of two high-energy bonds.

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h. Two other synthetic processes that generate PPᵢ

Examples:

• DNA synthesis (polymerization of nucleotides)

• RNA synthesis (transcription)

• Activation of fatty acids (formation of fatty acyl-CoA)

Significance of pyrophosphatase:

• PPᵢ + H₂O → 2 Pi rapidly occurs in cells.

• This drives biosynthetic reactions forward and makes them essentially irreversible by removing PPᵢ.

Discuss how Ile-tRNA synthetase distinguishes between Ile and Val at high fidelity

(p. 1020 - 1022).

How Ile-tRNA synthetase distinguishes Ile from Val (high fidelity)

• Isoleucine (Ile) and valine (Val) are very similar in structure (Val is slightly smaller), so the enzyme must ensure correct amino acid selection.

1. Initial selection (active site discrimination)

• Ile-tRNA synthetase active site is shaped to preferentially bind isoleucine.

• Because valine is slightly smaller, it can sometimes still fit into the active site and be activated.

2. Proofreading/editing mechanism

The enzyme has a second editing site that acts as a quality-control pocket.

Two possible corrections occur:

a. Pre-transfer editing

• If Val-AMP (valine attached to AMP) forms, the enzyme hydrolyzes it before transfer to tRNA.

b. Post-transfer editing

• If valine is mistakenly attached to tRNA (Val-tRNAᶦˡᵉ), the enzyme moves the tRNA to the editing site.

• The incorrect Val is hydrolyzed off the tRNA.

Double-sieve model

• First sieve (activation site): excludes larger amino acids than Ile.

• Second sieve (editing site): removes smaller amino acids like Val.

Result

• This double-sieve proofreading system ensures very high accuracy in attaching Ile to tRNAᶦˡᵉ and prevents incorporation of Val during protein synthesis.

What is the "second genetic code" (Fig. 27-21 [p. 1023] and p. 1022)

The “second genetic code” refers to the rules that determine how the correct amino acid is matched with its specific tRNA by aminoacyl-tRNA synthetases.

Key points:

• Aminoacyl-tRNA synthetases recognize specific structural features of each tRNA.

• These features include:

  • Anticodon sequence

  • Bases in the acceptor stem

  • Other identity elements in the tRNA structure

• These recognition signals ensure the correct amino acid is attached to the correct tRNA.

Importance:
This system guarantees that the codons in mRNA are translated into the correct amino acids during protein synthesis.

Use Fig. 27-24 (p. 1028) to discuss the key events in translation

initiation.

What is the Shine-Dalgarno sequence (p. 1028)? Use Fig. 27-25 (p. 1029) to

explain its significance in prokaryotic translation initiation.

Shine–Dalgarno sequence:

• A short purine-rich sequence in prokaryotic mRNA located ~5–10 nucleotides upstream of the start codon (AUG).

• Consensus sequence: AGGAGG.

Significance in translation initiation

• The Shine–Dalgarno sequence base-pairs with a complementary sequence in the 16S rRNA of the 30S ribosomal subunit.

• This aligns the ribosome correctly on the mRNA so that the start codon (AUG) is positioned in the P site.

• Proper positioning allows the initiator tRNA (fMet-tRNAᶠᴹᵉᵗ) to bind the start codon and begin translation.

Key idea:
The Shine–Dalgarno sequence recruits and positions the ribosome on prokaryotic mRNA for accurate initiation of protein synthesis.

Account for the cost of one high energy bond used when IF-2 delivers

an aminoacyl tRNA to the P site during translation initiation (p. 1028).

• IF-2 binds GTP and forms a complex with the initiator tRNA (fMet-tRNAᶠᴹᵉᵗ) and the 30S ribosomal subunit.

• IF-2 delivers the initiator aminoacyl-tRNA to the P site where it pairs with the start codon (AUG).

Energy cost:

• After correct assembly and binding of the 50S subunit, IF-2 hydrolyzes GTP → GDP + Pi.

• This GTP hydrolysis provides the energy required for:

  • Release of initiation factors

  • Proper assembly of the 70S initiation complex

✅ Thus, one high-energy phosphoanhydride bond from GTP is consumed during delivery of the initiator tRNA to the P site during translation initiation.

Compare the initiation of translation in prokaryotes and eukaryotes (pp. 1029 -

1030, and Figs. 27-26 and 27-27, p. 1030).

• Prokaryotes: ribosome is positioned by Shine–Dalgarno pairing.

• Eukaryotes: ribosome binds the 5′ cap and scans to the start codon with help of many initiation factors.

Feature	Prokaryotes	Eukaryotes
Ribosome	70S (30S + 50S)	80S (40S + 60S)
Initiator tRNA	fMet-tRNAᶠᴹᵉᵗ (N-formylmethionine)	Met-tRNAᵢᴹᵉᵗ (methionine)
mRNA recognition	Ribosome binds directly to Shine–Dalgarno sequence upstream of AUG	40S subunit binds 5′ cap and scans mRNA to find AUG
Positioning of start codon	Shine–Dalgarno sequence base-pairs with 16S rRNA to position AUG in P site	Scanning mechanism moves along mRNA until first suitable AUG
Initiation factors	IF1, IF2, IF3	Many eIFs (e.g., eIF1, eIF2, eIF3, eIF4E, eIF4G, eIF4A)
mRNA structure involved	Shine–Dalgarno region	5′ cap and poly(A) tail interact through proteins
mRNA organization	Often polycistronic (multiple proteins from one mRNA)	Usually monocistronic (one protein per mRNA)

Use Fig. 27-28 (p. 1031) to discuss the role of EF-Tu in

delivering AA-tRNAs to the A site during elongation.

Account for the one high energy bond used by EF-Tu.

What is the role of EF-Ts?

Role of EF-Tu in elongation:

• EF-Tu bound to GTP forms a complex with an aminoacyl-tRNA (aa-tRNA).

• This complex delivers the aa-tRNA to the A site of the ribosome.

• If the codon–anticodon pairing is correct, GTP is hydrolyzed → GDP + Pi.

• EF-Tu then releases the tRNA, allowing it to fully enter the A site.

Energy use:

• One high-energy phosphoanhydride bond is used when EF-Tu hydrolyzes GTP → GDP + Pi.

Role of EF-Ts:

• EF-Ts regenerates EF-Tu-GTP by exchanging GDP for GTP, allowing EF-Tu to participate in another cycle of aa-tRNA delivery.

Name the enzyme and point out the

nucleophilic attack mechanism catalyzed by

this enzyme. Is this reaction catalyzed by a

protein enzyme or a ribozyme?

2) Does protein synthesis run from N to C

terminal end -or- C to N?

3) Note the position of the peptidyl tRNA after

peptide bond formation.

4) Note: No additional input of energy is

required for peptide bond formation because

the required energy is released when the

peptidyl-tRNA ester bond is broken.

1. Enzyme and nucleophilic attack mechanism

• The enzyme activity is peptidyl transferase, located in the large ribosomal subunit (23S rRNA in bacteria).

• The reaction is catalyzed by rRNA, so the ribosome functions as a ribozyme (not a protein enzyme).

Mechanism:

• The α-amino group of the aminoacyl-tRNA in the A site acts as the nucleophile.

• It attacks the carbonyl carbon of the ester bond linking the growing polypeptide to the tRNA in the P site.

• This forms a peptide bond and transfers the growing peptide to the A-site tRNA.

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2. Direction of protein synthesis

• Protein synthesis proceeds from the N-terminus to the C-terminus (N → C).

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3. Position of the peptidyl-tRNA after peptide bond formation

• After the peptide bond forms, the growing polypeptide is attached to the tRNA in the A site (peptidyl-tRNA now in the A site).

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4. Energy requirement

• No additional energy input is required for peptide bond formation.

• The energy comes from the high-energy ester bond of the peptidyl-tRNA that is broken during the reaction.

Use Fig. 27-30 (p. 1034) to discuss translocation.

Account for the one high energy anhydride bond

that is used to translocate the peptidyl tRNA from

the A site to the P site?

Energy accounting

• One high-energy phosphoanhydride bond from GTP is hydrolyzed by EF-G.

• This GTP hydrolysis provides the energy for ribosome movement along the mRNA and tRNA repositioning.

Use Fig. 27-31 (p. 1035) to account for the one high

energy bond used in termination.

Folding and processing: Describe some ways that newly synthesized

polypeptides are processed following their synthesis (pp. 1036 - 1039).

1. Protein folding

• The polypeptide folds into its correct three-dimensional structure.

• Chaperone proteins (molecular chaperones) assist folding and prevent aggregation.

2. Proteolytic cleavage

• Some proteins are synthesized as inactive precursors (proproteins or zymogens).

• Specific peptide segments are removed to produce the active protein.

  • Example: proinsulin → insulin.

3. Covalent modifications

Proteins may undergo chemical modifications such as:

• Phosphorylation (addition of phosphate groups)

• Glycosylation (addition of carbohydrate groups)

• Acetylation or methylation

• Lipid attachment

These modifications can affect activity, stability, localization, or interactions.

4. Disulfide bond formation

• Cysteine residues form disulfide bonds that stabilize protein structure.

5. Assembly into multi-subunit complexes

• Some proteins associate with other polypeptides to form functional complexes (quaternary structure).

Key idea

These folding and modification steps convert the newly synthesized polypeptide into a stable, properly localized, and biologically active protein.

Consider a bacterial gene for a hypothetical polypeptide comprised of 100 amino

acid residues.

a. Approximately how many high-energy phosphate anhydride bonds are used to make the necessary

mRNA molecule that codes for this protein?

b. How many high-energy phosphate anhydride bonds are used to synthesize the hypothetical polypeptide

molecule from free amino acids, given the preformed mRNA?

c. Assuming that each mRNA molecule is translated 20-50 times, does the cell spend more energy in

transcription or translation?

a.

A 100-amino-acid protein needs about 100 codons + 1 stop codon = 101 codons
So the mRNA coding region is about:

• 101 × 3 = 303 nucleotides

Each nucleotide added during transcription uses the equivalent of 2 high-energy phosphate bonds (NTP → NMP + PPi, then PPi hydrolysis).

Approximate cost:

• 303 × 2 = ~606 high-energy bonds

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b.

For translation of a 100-aa polypeptide from free amino acids, approximate cost is:

• Amino acid activation: 100 × 2 = 200

• Delivery of aa-tRNA to A site: 99 × 1 = 99

• Translocation: 99 × 1 = 99

• Initiation + termination: about 2

Total ≈ 400 high-energy bonds

(Exam shortcut: ~4 high-energy bonds per amino acid, so 100 aa → ~400)

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c.

The cell spends more energy in translation.

Why:

• One mRNA costs about ~606 high-energy bonds to make.

• But one 100-aa protein costs about ~400 high-energy bonds to translate.

• If that mRNA is translated 20–50 times, translation costs:

  • 20 × 400 = 8000

  • 50 × 400 = 20,000

So translation >> transcription in total energy cost.

Many of the antibiotics used clinically are inhibitors of protein synthesis. Discuss why

some antibiotics are specifically used in the treatment of bacterial infections while

others are used to treat yeasts and fungi (pp. 1039 - 1041; Note: Mitochondrial and

bacterial ribosomes are similar and both are therefore sensitive to bacterial inhibitors.)

Bacterial antibiotics:

• Target 70S ribosomes (30S + 50S) found in bacteria.

• Human cells have 80S ribosomes, so these drugs mainly affect bacteria.

Yeast/fungal treatments:

• Yeasts and fungi are eukaryotes with 80S ribosomes, so different drugs are used that target fungal-specific features.

Note:

• Mitochondrial ribosomes are similar to bacterial ribosomes, so some bacterial antibiotics can affect mitochondria and cause side effects.

What is a "signal sequence" and what is its function

Signal sequence:

• A short sequence of amino acids at the N-terminus of a newly synthesized protein.

Function:

• Directs the protein to the correct cellular location (e.g., endoplasmic reticulum for secretion or membrane insertion).

• Often removed after the protein reaches its destination.