Molecular Biology of Translation: RNA Roles, Genetic Coding, and Ribosomal Mechanism, and Regulation

Introduction to Translation and RNA Roles

• Lecture seven focuses on the process of translation and is the first lecture covered on the second exam.
• Translation is the biological process where messenger RNA ($mRNA$) is used as a template to synthesize proteins.
• The primary RNAs involved in this process include:
  • Messenger RNA ($mRNA$): The template for protein synthesis.
  • Ribosomal RNA ($rRNA$): A structural and catalytic component of ribosomes.
  • Transfer RNA ($tRNA$): The adapter molecule that brings amino acids to the ribosome.
• Key components to be discussed include the genetic code, the structure and function of the ribosome, the three steps of translation (initiation, elongation, and termination), and the regulation of translation.

The Flow of Genetic Information and Cellular Compartmentalization

• The process begins with double-stranded DNA ($dsDNA$) in the nucleus of a eukaryotic cell.
• Transcription: $dsDNA$ undergoes transcription in the nucleus to produce RNA. This process uses ribonucleoside triphosphates ($rNTPs$) as building blocks.
• The sequence of the synthesized RNA is based on the sequence of the template strand of the DNA.
• Primary Transcript ($pre-mRNA$): The immediate product of transcription is a primary transcript, or $pre-mRNA$. Other RNAs like $rRNA$ and $tRNA$ are also synthesized as precursor versions.

RNA Processing and Nuclear Export

• Before becoming mature $mRNA$, the $pre-mRNA$ must undergo processing within the nucleus.
• Processing steps include:
  • Intron Removal: Excising non-coding regions called introns (often depicted in blue) and ligating exons (red regions) together.
  • $5'$ Capping: Adding a methyl-G cap to the $5'\text{ end}$.
  • $3'$ Polyadenylation: Adding a poly-A tail (a string of adenine nucleotides) to the $3'\text{ end}$.
• Mature $mRNA$ then undergoes nuclear export through nuclear pores in the nuclear envelope to enter the cytoplasm, where translation occurs.

Ribosome Assembly and the Nucleolus

• The nucleolus is a specialized site within the nucleus dedicated to:
  1. Transcription of $rRNA$ genes to synthesize $rRNA$.
  2. Assembly of newly synthesized $rRNA$ with ribosomal proteins to form ribosomal subunits.
• The two subunits are the large ribosomal subunit and the small ribosomal subunit.
• Once assembled, these subunits exit through nuclear pores into the cytoplasm to participate in translation along with $tRNA$, translation factors, and amino acids.

Nucleotide Structure and Biochemistry

• Nucleotides consist of three components:
  1. A nitrogenous base (nitrogen-containing base).
  2. A five-carbon sugar (pentose).
  3. A phosphate group.
• Definitions:
  • Nucleoside: The combination of a nitrogenous base and a sugar.
  • Nucleotide: A nucleoside with a phosphate group attached to the sugar ring.
• Sugar Types:
  • Ribose: Found in $RNA$; contains a hydroxyl group ($OH$) at the $2'\text{ carbon}$.
  • Deoxyribose: Found in $DNA$; the oxygen is lost at the $2'\text{ carbon}$ (replaced by hydrogen).

Nitrogenous Bases: Pyrimidines and Purines

• Pyrimidines (Single-ring structure):
  • The name is longer, but the structure is smaller (single ring).
  • Include Cytosine ($C$), Thymine ($T$), and Uracil ($U$).
  • Thymine is specific to $DNA$, while Uracil is specific to $RNA$.
  • They share the same ring structure but differ in functional groups (e.g., amino groups, methyl groups, or carbonyl groups).
• Purines (Double-ring structure):
  • The name is shorter, but the structure is larger (two rings).
  • Include Adenine ($A$) and Guanine ($G$).
  • Differences involve the placement of amino and carbonyl groups.

Nucleic Acid Directionality and Phosphodiester Bonds

• Nucleotides are coupled together via covalent bonds called phosphodiester bonds.
• A phosphodiester bond forms between the $3'\text{ OH}$ of one nucleotide and the $5'\text{ phosphate}$ of the next.
• Synthetic Precursors: Nucleotides must be in the triphosphate form ($dNTPs$ or $rNTPs$) for synthesis to occur.
• Example: A three-nucleotide sequence like $CAG$ maintains a free $5'\text{ end}$ at the $C$ and a free $3'\text{ end}$ at the $G$.
• Convention: Sequences are always written and read in the $5'$ to $3'$ direction.
• Example Notation: Deoxyguanosine $5'\text{ monophosphate}$ is abbreviated as $dGMP$ or simply $G$.

DNA Complementarity and Melting Temperature ($T_m$)

• Complementary Base Pairing:
  • $A$ pairs with $T$ (or $U$) via $2$\text{ hydrogen bonds}.
  • $G$ pairs with $C$ via $3$\text{ hydrogen bonds}.
• Anti-parallel Orientation: In $dsDNA$, one strand runs $5'$ to $3'$ and the other runs $3'$ to $5'$.
• Melting Temperature ($T_m$):
  • The temperature at which the two strands of DNA separate.
  • Single-stranded DNA ($ssDNA$) absorbs UV light at $260\,nm$ more efficiently than $dsDNA$.
  • $GC$ content is directly proportional to $T_m$. Since $G-C$ pairs have three bonds, they require more heat to separate than $A-T$ pairs.

Messenger RNA (mRNA) and Codons

• Codon: A non-overlapping group of three adjacent nucleotides in $mRNA$ that codes for a specific amino acid.
• Nature of the Code:
  • There are $4^3 = 64$ possible distinct codons.
  • $61$ codons encode amino acids.
  • $3$ codons are Stop Codons ($UAA, UAG, UGA$) which induce translational arrest and disassembly of the ribosome.
• Start Codon: $AUG$ encodes Methionine ($Met$) and serves as the translation start signal.
• Degeneracy: Since there are $20$ amino acids but $61$ coding codons, many amino acids are encoded by multiple codons (ranging from $2$ to $6$). Tryptophan ($UGG$) and Methionine ($AUG$) have only one codon each.

Reading Frames in Translation

• Every $mRNA$ contains three different potential reading frames.
• Each reading frame produces a completely different polypeptide sequence.
• Selection of the Correct Frame: The ribosome must identify the correct Start Codon ($AUG$) to establish the "Open Reading Frame" ($ORF$).
• An $ORF$ begins with a Start Codon ($AUG$) and ends with an in-frame Stop Codon.

Anatomy of a Mature Eukaryotic mRNA

• $5'\text{ Methyl-G Cap}$.
• $5'\text{ UTR}$ (Untranslated Region): Sequence between the cap and start codon.
• Open Reading Frame ($ORF$): The coding sequence.
• $3'\text{ UTR}$ (Untranslated Region): Sequence between the stop codon and the poly-A tail.
• $3'\text{ Poly-A Tail}$.

tRNA Charging and Aminoacyl tRNA Synthetases

• tRNAs must carry the correct amino acid to the ribosome. This is an enzyme-catalyzed reaction.
• Aminoacyl tRNA Synthetases: Enzymes that covalently couple a specific amino acid to its corresponding $tRNA$.
  • There are $20$ different synthetases (one for each amino acid).
  • Reaction Mechanism: The enzyme uses $ATP$, hydrolyzing it to $AMP$ and inorganic pyrophosphate.
  • Energy Bond: The amino acid is joined to the $3'\text{ end}$ of the $tRNA$ via a high-energy ester bond. This energy is later used to form the peptide bond during elongation.
• Charged tRNA: A $tRNA$ covalently coupled to its amino acid is called a "charged" or "aminoacyl" $tRNA$.

tRNA Structure and the Wobble Hypothesis

• Secondary Structure: Often called the "cloverleaf" structure.
  • $D\text{ loop}$: Contains dihydrouridine.
  • $T\psi C\text{ loop}$: Contains ribothymidine ($T$) and pseudouridine ($\psi$).
  • Anticodon loop: Contains the anticodon that base-pairs with the $mRNA$ codon.
  • Acceptor Arm: The $3'\text{ end}$ where the amino acid attaches.
• Tertiary Structure: The functional, three-dimensional L-shaped molecule.
• Wobble Position:
  • The first position ($5'\text{ position}$) of the anticodon is flexible.
  • Base Pairing Rules at Wobble Position:
    • $G$ can pair with $C$ or $U$.
    • $U$ can pair with $A$ or $G$.
    • Inosine ($I$), a modified base, can pair with $C, A,\text{ or } U$.
  • Significance: Allows cells to have fewer than $61$ distinct $tRNAs$ to recognize all amino acid-encoding codons.

Ribosome Composition and Architecture

• Ribosomes consist of $rRNA$ and proteins.
• Prokaryotic Ribosome ($70S$):
  • Large Subunit ($50S$): Includes $23S$ and $5S\text{ rRNAs}$ and $31$ proteins.
  • Small Subunit ($30S$): Includes $16S\text{ rRNA}$ and $21$ proteins.
• Eukaryotic Ribosome ($80S$):
  • Large Subunit ($60S$): Includes multiple larger $rRNAs$ (including $28S, 5.8S, 5S$) and $50$ proteins.
  • Small Subunit ($40S$): Includes $18S\text{ rRNA}$ and $33$ proteins.
• Ribosomal Sites:
  • A Site (Aminoacyl site): Holds the incoming charged $tRNA$.
  • P Site (Peptidyl site): Holds the $tRNA$ carrying the growing polypeptide chain.
  • E Site (Exit site): Holds uncharged $tRNAs$ before they are released.

Eukaryotic Initiation of Translation

• Initiation Factors ($eIFs$) keep subunits apart and assist in identifying the $5'\text{ cap}$.
• Methionine tRNAs: Cells have two distinct $tRNAs$ for Methionine:
  • $tRNA_i^{Met}$: Specifically for initiation.
  • $tRNA^{Met}$: For internal methionine incorporation during elongation.
• Initiation Process:
  1. $eIF2-GTP$ binds to $tRNA_i^{Met}$ and brings it to the $P\text{ site}$ of the $40S$ subunit to form the $43S$ pre-initiation complex.
  2. The $eIF4$ complex (consisting of $4E, 4G, 4A,\text{ and } 4B$) recognizes the $5'\text{ cap}$ of the $mRNA$.
  3. $eIF4E$ binds to the cap; $eIF4G$ interacts with Poly-A Binding Protein ($PABP$) on the tail to circularize/verify the $mRNA$.
  4. The $43S$ complex binds to the $mRNA$ and scans from $5'$ to $3'$ searching for the first $AUG$.
  5. Identification of $AUG$ causes $eIF2-GTP$ hydrolysis ($eIF2-GDP$), forming the $48S$ complex.
  6. Most $eIFs$ fall off; the $60S$ subunit joins with help from $eIF5B-GTP$. Upon hydrolysis, $eIF5B$ and $eIF1A$ are released, leaving a mature $80S$ ribosome.

Translation Elongation and Peptidyltransferase

• Elongation Factors ($EEFs$) facilitate the process.
• Elongation Cycle:
  1. $EEF1A-GTP$ brings a charged $tRNA$ to the vacant $A\text{ site}$. If correct, $GTP$ is hydrolyzed.
  2. Peptidyltransferase Reaction: A peptide bond forms between the amino acid in the $P\text{ site}$ and the one in the $A\text{ site}$. Methionine is transferred from its $tRNA$ to the new amino acid in the $A\text{ site}$.
  3. The enzyme is a ribozyme (catalytic activity comes from the $rRNA$).
  4. Translocation: $EEF2-GTP$ causes the ribosome to move one codon down. The uncharged $tRNA$ moves from $P\text{ to } E$, and the peptidyl-$tRNA$ moves from $A\text{ to } P$.

Translation Termination

• Termination begins when a stop codon ($UAA, UAG,\text{ or } UGA$) enters the $A\text{ site}$.
• No $tRNA$ recognizes stop codons; instead, Eukaryotic Release Factors ($eRFs$) bind.
• $eRF1$ recognizes the stop codon.
• $eRF3-GTP$ provides energy to cleave the polypeptide from the last $tRNA$.
• The entire complex disassembles, and ribosomal subunits are recycled.

Polysomes and Translational Efficiency

• Polysomes (Polyribosomes): A single $mRNA$ molecule being translated by multiple ribosomes simultaneously.
• This structure increases the efficiency of protein synthesis.
• As one ribosome moves down the $mRNA$, another can initiate at the $5'\text{ end}$.
• A "monosome" refers to an $mRNA$ translated by only a single ribosome.

Regulation and Antibiotics

• Regulating Initiation: $eIF2$ is a critical control point.
  • $eIF2$ must exchange $GDP$ for $GTP$ to be reactivated.
  • Phosphorylation of $eIF2$ blocks this exchange, keeping it in an inactive $GDP$-bound state, thereby arresting translation.
• Antibiotics: Many antibiotics target specific steps in translation:
  • Binding to $A\text{ or } P$ sites to affect decoding.
  • Blocking $tRNA$ binding to the $A\text{ site}$.
  • Inhibiting translocation.
  • These often show selectivity for prokaryotic ($70S$) vs eukaryotic ($80S$) ribosomes.