Ch. 39 Genetic Code

Administrative Details and Lecture Introduction

• Students are assured of receiving an extra five points, which equates to slightly more than $1\%$. This is described as a beneficial addition to their grade.

• Students who have not yet filled out course evaluations are encouraged to do so, as feedback is useful for course planning. Evaluations remain open through Saturday.

• The current lecture is part of a pair focused on translation. Previous lectures discussed "preparing the road for ribosomal traffic" by manufacturing messenger RNA (mRNA).

• In eukaryotes, this involved adding features such as on-ramps and smooth curves (metaphorical descriptions of post-transcriptional modifications) to ensure good motility of ribosomes along the mRNA template.

• The focus of the current discussion is the interpretation of nucleic acid sequences via the genetic code, while upcoming lectures will focus on the charging of transfer RNAs (tRNAs).

Historical and Cultural Context of Translation

• Saint Jerome (early 4th century) is cited as perhaps the most famous translator. He traveled from Dalmatia to the Near East to access original Hebrew texts for the Bible's Latin translation (the Vulgate), rather than relying on Greek intermediaries.

• A painting by Caravaggio (early 1600s) depicts Saint Jerome, jokingly noted by the speaker for his "good home gym" physique in the artwork.

• A contemporary icon of translation mentioned is the Duolingo mascot, illustrating how visualizations of translation have evolved while the core concept remains the same.

Defining the Genetic Code

• The genetic code is the mechanism by which a nucleic acid sequence is converted into a sequence of amino acids in proteins.

• Nucleic acids and amino acids are not a natural chemical match, requiring encoded information in DNA to be generated into an RNA copy and then into meaningful protein sequences.

• Key features of the code include:     * Triplets: Three nucleotides (bases) are required to specify one single amino acid.     * Non-overlapping: Each group of three nucleotides is used only for its own triplet. The code does not "backtrack" to use the last element of one triplet as the first of the next.     * Lack of Punctuation: There is no surplus material or nucleotide indicating a space, period, semicolon, or comma between amino acids. The code is a continuous run of consecutive amino acids.     * Directionality: The code must be read in a specific direction ($5'$ to $3'$).     * Degeneracy: The code is redundant, meaning multiple different triplets can specify the same amino acid. This is compared to degenerate orbitals in chemistry rather than moral degeneracy.

Structure and Usage of the Genetic Code Table

• The code was determined in the 1960s through arduous labor involving synthetic RNA runs (e.g., poly-U sequences to identify the code for Phenylalanine).

• The table consists of 64 possible triplets ($4 \times 4 \times 4 = 64$).

• Standard Table Organization:     * First Position (First Base): Dictates the large blocks of four rows.     * Second Position (Second Base): Dictates the specific column within the block.     * Third Position (Third Base): Dictates the specific row.

• Example: A sequence of $CUG$ specifies the amino acid Leucine ($Leu$).

• Out of 64 possibilities, 61 specify amino acids. The remaining three are special "Stop" codons:     * $UAA$     * $UAG$     * $UGA$

• When translation arrives at these triplets, the assembly of the protein is terminated.

Advantages of Redundancy and Degeneracy

• Error Buffering: Redundancy provides a safeguard against mutations. For example, if a mutation occurs in the third position of a Leucine triplet, there is a high probability it will still specify Leucine.

• Minimal Length Logic: Since there are 20 canonical amino acids, a single base ($4$ possibilities) or two bases ($4 \times 4 = 16$ possibilities) is insufficient. Triplets provide $64$ possibilities, which is the minimally sufficient length despite being "excessive."

• Prevention of Premature Termination: It is more advantageous to fill the "excess" 44 slots with amino acids rather than stop codons. A mutation resulting in a stop codon is often "damning" as it creates a non-functional, truncated protein (e.g., 200 amino acids instead of 1000).

• Universality: The code is nearly universal across all life forms ($E. coli$ uses the same code as humans). This allows for the manufacturing of biologics, such as producing human insulin in bacteria for Type 1 diabetes patients.

Exceptions to the Universal Genetic Code

• Some organisms like ciliate protozoa have slight variations in stop codon usage.

• Mitochondria: Human mitochondria possess their own genomes, tRNAs, and ribosomes (different from cytosolic ones). They interpret some codons differently:     * $UGA$: Specified as Stop in the universal code; specifies Tryptophan ($Trp$) in mitochondria.     * Arginine Triplets ($AGG, AGA$): Specified as amino acids in the universal code; specify Stop in mitochondria.

• Cross-translation Consequences:     * If a cytoplasmic mRNA sequence containing $UGA$ is translated in the mitochondria, it will result in an "overgrown" protein because the stop signal is ignored.     * Conversely, shifting mitochondrial sequences to the cytoplasm can result in proteins that are too short due to premature stop recognition.

Interactive Exercise: Code Breaking

• The speaker provides a sample message in triplets for translation.

• The decrypted message is revealed to be: "Sadly the yak's milk smells."

Key Players in Translation: tRNA and Synthetases

• Transfer RNAs (tRNAs): The critical bridge nucleic acids that bring amino acids to the ribosome via hydrogen bonding with mRNA.

• Aminoacyl tRNA Synthetases: Enzymes that "charge" tRNAs by attaching the correct amino acid. A tRNA without its amino acid cargo is useless for translation.

• tRNA Structure:     * 2D Structure: Clover-like structure.     * $DHU$ Loop: Contains Dihydrouridine (uracil ring with double bond removed and two additional hydrogens).     * $T\Psi C$ Loop: Contains Ribothymidylate ($T$), Pseudouridine ($\Psi$), and Cytosine ($C$).     * $3'$ Terminus: Always ends in a $CCA$ sequence; this is the point of amino acid attachment.     * Anticodon Loop: Contains the three-nucleotide sequence that interacts with mRNA.     * 3D Structure: Resembles a Greek "gamma" or a drill, with the anticodon loop and $3'$ terminus exposed at opposite ends.

• Modified Bases: tRNAs include cytosine, methylated versions of cytosine, and Inosine (base: hypoxanthine). These modifications allow for precise folding and accurate identification by synthetases.

tRNA-mRNA Interactions and Wobble

• Interactions occur via an anti-parallel arrangement: The mRNA reads $5'$ to $3'$, while the tRNA anticodon pairs $3'$ to $5'$ ($5'$ to $3'$ in the opposite direction).

• Convention: tRNA sequences are always recorded in the $5'$ to $3'$ direction.

• Example: A tRNA for Tryptophan ($Trp$) with an anticodon $CCA$ ($5' \rightarrow 3'$) pairs with the mRNA codon $UGG$ ($5' \rightarrow 3'$).

• Wobble Hypothesis: There are 61 codons but fewer types of tRNA in most organisms. tRNAs can read more than one codon through "wobble" at the first position of the anticodon (which corresponds to the third position of the codon).

• Wobble Rules:     * The first two bases of the mRNA codon must pair exactly (canonical Watson-Crick base pairing: $A-U, C-G$).     * If the first base of the anticodon is $C$: Pairs only with $G$.     * If the first base of the anticodon is $A$: Pairs only with $U$.     * If the first base of the anticodon is $U$: Can pair with $A$ or $G$.     * If the first base of the anticodon is $G$: Can pair with $C$ or $U$.     * If the first base of the anticodon is Inosine ($I$): Can pair with $U$, $C$, or $A$.

• Efficiency: An Alanine tRNA with anticodon $IGC$ can read three different codons ($GCU, GCC, GCA$).

Charging tRNAs: Aminoacyl tRNA Synthetases

• Attachment occurs at the $3'$ (and sometimes initially $2'$) hydroxyl group of the terminal adenosine of the $CCA$ tail.

• Reaction mechanism:     1. Adenylation: Amino Acid + $ATP \rightarrow$ Aminoacyl-AMP ($adenylate$) + $PP_i$ (pyrophosphate).     2. Pyrophosphate Hydrolysis: $PP_i$ is split into two molecules of inorganic phosphate ($2 P_i$), driving the reaction forward.     3. Transfer: The aminoacyl group is transferred from AMP to the tRNA.

• Energetics: The process costs one $ATP$ molecule, but because it is hydrolyzed to $AMP$, it is the energy equivalent of consuming two $ATP$ molecules ($ATP \rightarrow AMP + 2 P_i$).

• Accuracy and Proofreading: Ribosomes cannot check if the correct amino acid is attached to a tRNA. Synthetases must ensure accuracy.

• Double Sieve Mechanism (Example: Threonine Synthetase):     1. Active Site Sieve: Uses a coordination Zinc ($Zn^{2+}$) ion to interact with the polar hydroxyl group of Threonine ($Thr$). It excludes Valine ($Val$) because Valine lacks the polar group. However, Serine ($Ser$) also has a polar hydroxyl and can occasionally be mis-incorporated.     2. Editing Site Sieve: The editing site is smaller than the active site. Threonine is too bulky to enter the editing site, but the smaller, mis-incorporated Serine enters and is hydrolyzed off the tRNA.

• Synthetase Recognition: Synthetases "drape" around tRNAs, interpreting the anticodon loop and other structural features. Different synthetases (e.g., Phenylalanine vs. Threonine) vary significantly in size and shape.

Error Rates and Translational Control

• Error Rates: The average protein in $E. coli$ is 300 amino acids long.     * If the error rate is $1/100,000$, approx $99\%$ of proteins are correct.     * If the error rate is $1/100$, almost no long proteins (1000 amino acids) will be functional.     * Typical biological error rates fall between $1/10,000$ and $1/100,000$.

• Codon Usage Bias: Redundancy allows for translational speed control.     * Abundant tRNAs allow for fast translation and high initiation rates.     * Rare tRNAs slow down translation ("gum up the works").     * Purpose of slowing: In multi-domain proteins, rare codons can provide time for one domain to fold properly before the next is synthesized.

• Experimental Modification: Scientists can create "jazzed up" tRNAs with labels or eliminate synonymous codons from genomes to make bacteria immune to viruses (since the virus relies on the standard code).

Ribosomes and Co-transcriptional Translation

• Ribosomes are enzymes made of RNA and protein (ribozymes).

• Bacterial Ribosome ($E. coli$):     * Large Subunit ($50S$): Contains various RNAs and proteins.     * Small Subunit ($30S$): Contains various proteins and the essential ribosomal RNA.

• Ribosomal RNA is highly repetitive; a single $E. coli$ cell may contain $100,000$ copies of ribosomes during rapid growth.

• In bacteria, translation and transcription occur simultaneously (co-transcriptional translation). Ribosomes attach to nascent mRNA while it is still being synthesized by RNA Polymerase.

Attenuation: The Tryptophan ($trp$) Operon

• Attenuation is a regulatory process where gene expression is controlled by the speed of translation.

• The $trp$ operon contains genes for synthesizing Tryptophan. It is needed when Tryptophan levels are low.

• Mechanism depends on mRNA secondary structure (competing stem-loops):     * Region 1: Contains two back-to-back Tryptophan codons.     * High Tryptophan: The ribosome moves quickly through the leader sequence, covering region 2. This allows regions 3 and 4 to form an intrinsic terminator (hairpin loop). Transcription terminates prematurely.     * Low Tryptophan: The ribosome stalls at the Tryptophan codons in region 1, waiting for rare $tRNA^{Trp}$. This allows region 2 to pair with region 3 (anti-terminator loop). The 3-4 terminator loop cannot form, allowing RNA polymerase to continue transcribing the structural genes.