Gene Expression II: The Genetic Code and Protein Synthesis (Translation)

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Gene Expression II: The Genetic Code and Protein Synthesis

Introduction to Gene Expression and Translation

Gene expression is the process by which information from a gene is used to synthesize a functional gene product, often a protein. For many genes, the final product is a protein, and the instructions for assembling amino acids into a polypeptide are encoded in messenger RNA (mRNA). Translation is the cellular process that interprets the genetic code in mRNA to build proteins.

RNA transcript: For some genes, the RNA itself is the final product (e.g., rRNA, tRNA).
mRNA: Encodes instructions for translation, specifying the sequence of amino acids in a polypeptide.
Translation: The process of assembling amino acids into a polypeptide chain based on mRNA sequence.

The Mechanism of Translation

Key Learning Objectives

Describe the role of the ribosome in translation.
Explain the function of aminoacyl-tRNA in translation.
Explain the role of messenger RNA (mRNA) in translation.
Understand the roles of initiation factors, elongation factors, and release factors.
Define key terms: ribosome, tRNA, shine-dalgarno, A-site, P-site, E-site, mRNA, start codon, stop codon.

Translation: The Cast of Molecular Players

Major Components

Ribosomes: Carry out polypeptide synthesis by facilitating the assembly of amino acids into proteins.
tRNA molecules: Align amino acids in the correct order by matching their anticodon to the mRNA codon.
Aminoacyl-tRNA synthetases: Enzymes that attach amino acids to their corresponding tRNA molecules.
mRNA molecules: Encode the amino acid sequence information.
Protein factors: Facilitate steps of translation, including initiation, elongation, and termination.

Ribosomes: Structure and Function

General Properties

Ribosomes are complexes of ribosomal RNA (rRNA) and protein.
In eukaryotes, ribosomes are found free in the cytoplasm or bound to the endoplasmic reticulum (ER) and the outer nuclear envelope.
In prokaryotes, ribosomes are smaller and found free in the cytoplasm.

Ribosome Structure

Ribosomes are built from two dissociable subunits: large subunit and small subunit.
Bacterial ribosomes are sensitive to different inhibitors of protein synthesis and are composed of fewer proteins and rRNA molecules than eukaryotic ribosomes.

Properties of Bacterial and Eukaryotic Cytoplasmic Ribosomes

The following table compares the main properties of bacterial and eukaryotic ribosomes:

Organism	Ribosome Size (S Value)	Large Subunit (S Value)	Small Subunit (S Value)	Subunit Proteins	Subunit rRNA
Bacteria	70S	50S	30S	Large: 34 Small: 21	Large: 23S, 5S Small: 16S
Eukaryotes	80S	60S	40S	Large: ~46 Small: ~32	Large: 28S, 5.8S, 5S Small: 18S

Additional info: S value (Svedberg unit) is a measure of sedimentation rate during centrifugation, reflecting size and shape.

Ribosome Sites Important for Protein Synthesis

mRNA-binding site: Where mRNA binds to the ribosome.
A (aminoacyl) site: Binds incoming aminoacyl-tRNA (charged tRNA with attached amino acid).
P (peptidyl) site: Holds the tRNA carrying the growing polypeptide chain.
E (exit) site: Where tRNA exits the ribosome after releasing its amino acid.

Transfer RNA (tRNA) and Aminoacyl-tRNA Synthetases

tRNA Structure and Function

tRNA is an adaptor molecule that binds both a specific amino acid and the mRNA codon specifying that amino acid.
Each tRNA is linked to its amino acid by an ester bond and named for the attached amino acid (e.g., tRNAAla for alanine).
tRNAs attached to amino acids are called aminoacyl-tRNAs (charged tRNAs), and the amino acid is considered activated.
tRNA recognizes codons in mRNA via complementary base pairing between its anticodon and the mRNA codon.

Wobble Hypothesis

The wobble hypothesis explains flexibility in base pairing between the third base of the mRNA codon and the corresponding base of the tRNA anticodon.
This allows some tRNAs to recognize more than one codon, reducing the number of tRNAs required.
Inosine (I), formed by RNA editing from adenosine, often occurs at the wobble position and can pair with U, C, or A.
Example: tRNA with anticodon 3'-UAI-5' can recognize codons AUA, AUC, and AUU (all coding for isoleucine).

Aminoacyl-tRNA Synthetases

Before tRNA can deliver an amino acid to the ribosome, the amino acid must be covalently attached by an aminoacyl-tRNA synthetase.
Cells have 20 different aminoacyl-tRNA synthetases, one for each amino acid.
Some cells with nontraditional amino acids have special tRNAs and synthetases.

Reaction Catalyzed by Aminoacyl-tRNA Synthetase

The attachment of an amino acid to tRNA (amino acid activation) is catalyzed by aminoacyl-tRNA synthetase and requires ATP hydrolysis:

General reaction:

Both the anticodon and the 3' end of tRNA are necessary for correct amino acid attachment.
Synthetases proofread the final product to ensure accuracy.

Messenger RNA (mRNA) and Its Role in Translation

mRNA Structure and Function

The sequence of codons in mRNA determines the order of amino acids in the polypeptide.
mRNA must be exported from the nucleus to the cytoplasm in eukaryotes.
Untranslated regions (UTRs) at the 5' and 3' ends are essential for mRNA function.
Start codon (usually AUG) signals the beginning of translation; stop codons (UAG, UAA, UGA) signal termination.
mRNAs have a 5' cap and a 3' poly(A) tail in eukaryotes.

Comparison of Prokaryotic and Eukaryotic mRNA

Most eukaryotic mRNAs are monocistronic (encode one polypeptide).
Bacterial and archaeal mRNAs can be polycistronic (encode several polypeptides, often with related functions).
Polycistronic transcription units are called operons.

Protein Factors in Translation

Initiation, Elongation, and Termination Factors

Each stage of translation requires specific protein factors:
- Initiation factors: Assist in the assembly of the ribosome and mRNA.
- Elongation factors: Facilitate the addition of amino acids to the growing chain.
- Release factors: Recognize stop codons and trigger termination.

Mechanism of Translation

Overview of Translation

Translation begins at the N-terminus of the polypeptide and proceeds to the C-terminus.
mRNA is read in the 5' to 3' direction.
Translation is divided into three stages:
1. Initiation: mRNA and initiator tRNA bind to the ribosome and are positioned for translation.
2. Elongation: Amino acids are sequentially joined via peptide bonds.
3. Termination: mRNA and the polypeptide are released from the ribosome.

Initiation of Translation

Bacterial Initiation

Three initiation factors (IF1, IF2, IF3) bind to the small (30S) ribosomal subunit; IF2 binds GTP.
mRNA binds to the small subunit, oriented by the Shine-Dalgarno sequence (purine-rich region upstream of the start codon).
Initiator tRNA carrying N-formylmethionine (fMet) binds the P site.
The 30S initiation complex forms, then binds the large (50S) subunit to create the 70S initiation complex.
GTP hydrolysis releases initiation factors.

Eukaryotic Initiation

Start codon specifies methionine (not N-formylmethionine).
Initiation factors are called eIFs (about a dozen).
Initiator tRNA binds eIF2-GTP, then the small ribosomal subunit and other eIFs to form the 43S preinitiation complex.
mRNA 5' cap is recognized by eIF4E, which recruits eIF4G and other factors.
The small subunit scans mRNA for the first AUG, often within a Kozak sequence (e.g., ACCAUGG).
Large subunit joins after start codon recognition; GTP hydrolysis releases eIFs.
Poly(A)-binding protein (PABP) at the 3' end interacts with eIF4G, stabilizing the mRNA.
Some mRNAs use an internal ribosome entry sequence (IRES) for ribosome recruitment.

Elongation

Elongation involves cycles of aminoacyl-tRNA binding, peptide bond formation, and translocation.
Incoming aminoacyl-tRNA binds the A site, assisted by elongation factors (EF-Tu, EF-Ts in bacteria) and GTP hydrolysis.
Peptide bond forms between the amino group of the A site amino acid and the carboxyl group of the P site amino acid.
The growing peptide chain is transferred to the tRNA in the A site.
Translocation moves the peptidyl-tRNA from the A site to the P site, and the empty tRNA to the E site, facilitated by EF-G and GTP hydrolysis.
Translation error rate is low (about 1 in 10,000).

Peptide Bond Formation

Peptidyl transferase activity is catalyzed by rRNA (23S in bacteria), making the ribosome a ribozyme.

Polyribosomes (Polysomes)

Multiple ribosomes can translate a single mRNA simultaneously, forming a polyribosome or polysome.
This increases the efficiency of protein synthesis.

Termination

Translation ends when a stop codon (UAG, UAA, UGA) enters the A site.
Stop codons are recognized by release factors, not tRNAs.
Release factors mimic tRNA structure and bind the A site, triggering release of the polypeptide from the ribosome.
GTP hydrolysis facilitates the dissociation of the translation complex.

Summary Table: Key Terms in Translation

Term	Definition
Ribosome	Macromolecular complex of rRNA and protein that synthesizes polypeptides
tRNA	Adaptor molecule that brings amino acids to the ribosome
mRNA	Messenger RNA; encodes the sequence of amino acids
Shine-Dalgarno sequence	Purine-rich sequence in prokaryotic mRNA that helps position the ribosome
Start codon	First codon translated (usually AUG)
Stop codon	Codon that signals termination (UAG, UAA, UGA)
A site	Aminoacyl site; binds incoming aminoacyl-tRNA
P site	Peptidyl site; holds tRNA with growing polypeptide
E site	Exit site; where tRNA leaves the ribosome