Genetic Code - Lecture 39 Review

BMB 3110 Lecture 39 Notes

Chapter 39: The Genetic Code Outline

• Characteristics of the code

• tRNA structure

• Pairing and wobble

• Aminoacyl-tRNA synthetases

• Ribosomes

• See problems 1-5, 9-10, and 12-17 at the end of Chap. 39

Lecture 39: Learning Goals

At the end of this lecture, you should:

• Understand general properties of the genetic code

• Know shared structural features of tRNAs

• Be able to describe how interactions between tRNA and mRNA specify amino acids

• Know the function of tRNA synthetases

• Understand basic principles of proofreading/editing in tRNA charging

• Be familiar with co-transcriptional translation in bacteria

Properties of the Genetic Code

• The genetic code connects the sequence of nucleic acids in DNA (or RNA) to the sequence of amino acids in proteins. It enables the utilization of information stored in nucleic acid sequences.

• Characteristics of the genetic code:

  • It consists of triplets (codons): Each codon consists of three nucleotide bases.

  • It is non-overlapping: The code is read sequentially without overlap between codons.

  • It does not include punctuation: There are no special symbols to mark the boundaries of codons.

  • It has 5’-to-3’ directionality: Codons are read in the direction from the 5’ end to the 3’ end.

  • It is degenerate: More than one codon can code for the same amino acid.

Introducing the Genetic Code

• A critical feature of the code is that not all codons specify amino acids.

  • There are three stop codons: UAA, UAG, and UGA.

  • Many amino acids are represented by more than one codon.

Universality of the Genetic Code

• Most organisms utilize an identical genetic code.

  • The genetic code is nearly universal, which allows for the production of recombinant proteins such as insulin in bacteria.

  • Small differences in the code have been identified:

  • In ciliated protozoa: UGA functions as one stop codon instead of three.

  • In mitochondria: Four stop codons present; this variation exists because mitochondria have their own tRNAs (McFarland et al. 2004).

Table 39.1: The Genetic Code

Key Players in Protein Synthesis

Transfer RNAs (tRNAs)

• tRNAs are essential for linking mRNA with specific amino acids.

• They contain approximately 70-90 nucleotides and have a characteristic L-shaped three-dimensional structure.

• Secondary structure of tRNAs includes five unpaired regions, including:

  • Acceptor stem: contains the 3’ CCA terminus.

  • Anticodon loop: responsible for pairing with mRNA codons.

• Mature tRNAs include modified bases, such as inosine, methylated cytosine, and dihydrouridine.

tRNA-mRNA Interactions

• Each codon on an mRNA base pairs with the complementary anticodon on tRNA.

  • The direction of translation is 5’-to-3’ along mRNA.

  • tRNA nomenclature: tRNA is named based on its anticodon or the amino acid it carries. Example: tRNA^Trp (for tryptophan) corresponds to the anticodon sequence CAU.

Wobble in tRNA-mRNA Interactions

• There are 61 codons that specify amino acids, but there are fewer than 61 unique tRNAs.

  • This implies that some tRNAs can recognize multiple codons (wobble phenomenon).

  • The last two bases of the anticodon pair precisely with the corresponding bases in the mRNA codon.

  • The first base of the anticodon determines how many codons it can recognize:

  • If the first base is C or A: it pairs with one specific codon.

  • If U or G: it pairs with two codons.

  • If I (inosine): it can pair with three different codons.

• Example: tRNA^I_GC can read the codons GCU, GCC, and GCA (encoding alanine).

Preparing tRNAs for Translation

• Before tRNAs can participate in translation, they must be charged with the correct amino acid.

  • This charging process is referred to as the formation of aminoacyl-tRNA, which includes an ester bond at the 3’ CCA arm. The bond can occur at either the 2’ or 3’-OH of the ribose.

• The steps for producing charged tRNAs entail:

  • Activation of the amino acid.

  • Transfer of the activated amino acid to the tRNA.

  • Proofreading of the newly added tRNA to ensure specificity.

Activating Amino Acids

• Activation requires the amino acid to react with ATP to form aminoacyl adenylate, which releases pyrophosphate.

• The aminoacyl adenylate (AMP) is subsequently transferred to the tRNA.

• Both activation and transfer steps are catalyzed by aminoacyl-tRNA synthetases, resulting in a two ATP equivalent cost for this process.

Selecting the Right Amino Acid

• Aminoacyl-tRNA synthetases must exhibit high specificity to distinguish between similar amino acids.

• Example with threonine:

  • Threonine has a hydroxyl group that forms specific interactions within the active site of the synthetase preventing the addition of structurally similar valine.

  • Editing sites also play a critical role in rejecting incorrectly added amino acids, ensuring accuracy during tRNA charging.

Selecting the Right tRNA

• Synthetases match the appropriate amino acids with the correct tRNAs, in addition to relying on the anticodon loop, other sequence regions with modified bases are often utilized for recognition.

Error Rates in Protein Synthesis

• Errors can occur due to incorrect tRNA-amino acid coupling or mispairing during codon-anticodon interactions.

• To maintain protein functionality, errors must be infrequent.

• The frequency of inserting an incorrect amino acid across different sequences shows exponential decay; an incorrect addition might be on the order of:

  • 100 amino acids - $10^{-2}$ error rate

  • 300 amino acids - $0.366$

  • 1000 amino acids - $0.0001
    ightarrow 10^{-4}$

• The actual error rate in peptide synthesis is between $10^{-5}$ and $10^{-4}$.

Consequences of Degeneracy: Codon Bias

• Degeneracy in the genetic code offers advantages, acting as a buffer against mutations and allowing for translational control through various tRNA abundances.

  • The selection among synonymous codons can influence translational efficiency.

  • Reference for this topic includes Hanson and Coller (2018).

Altering the Genetic Code

• An understanding of the genetic code provides the foundation for systematic modifications, including:

  • Addition of custom amino acids for specific functions such as fluorescence or labeling.

  • Elimination of synonymous codons to confer viral resistance (Kwon 2023).

Building Proteins: Ribosomes

• Ribosomes are complexes that enable the translation of mRNAs utilizing charged tRNAs.

• The E. coli ribosome is composed of two subunits:

  • 50S (large) subunit: consists of 34 proteins (L1-L34) and rRNA (23S and 5S).

  • 30S (small) subunit: includes 21 proteins (S1-S21) and 16S rRNA.

• Ribosomes are ribozymes, with rRNA accounting for approximately two-thirds of their total mass.

• Ribosomes are abundant, with up to 100,000 copies per cell.

Co-Transcriptional Translation in E. coli

• The direction of translation corresponds to the direction of mRNA synthesis, which is always from 5’ to 3’.

• Ribosomes can engage in co-transcriptional translation when they have access to mRNA.

Bacterial Transcription Regulation: Attenuation

• The trp operon, essential for tryptophan synthesis, is regulated through attenuation.

• It modulates transcription based on tryptophan levels:

  • High tryptophan concentrations allow ribosomes to translate through tryptophan-coding codons, leading to stem-loop formation in mRNA.

  • Low tryptophan levels cause ribosomes to stall, resulting in alternative mRNA secondary structures that do not form stem-loops.

Key Concepts Recap

• General properties of the genetic code:

  • Important features, including degeneracy and universal tRNA characteristics.

• Charging tRNA with amino acids and the necessary steps.

• The interaction of tRNA with mRNA, including wobble configurations.

• The distinctive characteristics allowing for versatility and errors in translation processes to ensure functional proteins.