BMB 3110 Lecture 39: The Genetic Code

BMB 3110 Lecture 39: The Genetic Code

Lecture Outline:

  • Characteristics of the genetic code

  • tRNA structure

  • Pairing and wobble

  • Aminoacyl-tRNA synthetases

  • Ribosomes

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

Learning Goals:

At the end of this lecture, you should:

  • Understand general properties of the genetic code

  • Know shared structural features of tRNAs

  • 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

The Genetic Code:

  • The genetic code connects the sequence of nucleic acids in DNA (RNA) to the sequence of amino acids in proteins.

  • It enables the use of information stored in nucleic acid sequences.

Characteristics of the Genetic Code:

  • Triplet Code: The genetic code consists of triplets, which are sequences of three nucleotides.

  • Nonoverlapping: The code is read sequentially without overlapping. Each nucleotide is part of only one codon.

  • No Punctuation: The code does not contain punctuation marks that could interrupt the reading frame.

  • Directionality: The code has a specific direction that is read from 5’-to-3’.

  • Degeneracy: The genetic code is degenerate, meaning that multiple codons can specify the same amino acid.

Critical Features of the Genetic Code:

  • 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 (e.g., Leucine, Serine).

Universality of the Genetic Code:

  • Most organisms use an identical genetic code, contributing to its nearly universal nature.

  • This universality allows for instances such as the production of insulin in bacteria.

  • Some small differences are known:

    • Ciliated Protozoa: One stop codon (UGA) only.

    • Mitochondria: Four stop codons, functioning with their own tRNAs.

The Genetic Code Table (Simplified):

  • Amino acids and their corresponding codons are listed, detailing:

    • 3-Letter, 1-Letter abbreviation,

    • Positions for each nucleotide base in codons

    • Example:

    • Alanine (Ala, A):

      • First position can be U, C, A, G

      • Second position can be U, C, A, G

      • Third position can also vary.

Transfer RNAs (tRNAs):

  • tRNAs are single-stranded RNAs, approximately 70-90 nucleotides in length.

  • They adopt an L-shaped 3D structure.

Structure of tRNAs:

  • Secondary Structure includes five unpaired regions:

    • Acceptor stem (includes 3’ CCA terminus)

    • Anticodon loop

  • Mature tRNAs contain many modified bases, such as:

    • Inosine

    • Methylated cytosine

    • Dihydrouridine

tRNA-MRNA Interactions:

  • Each codon on mRNA pairs with a complementary anticodon on tRNA.

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

  • tRNA is named based on its anticodon.

    • Example: tRNATrp (equivalent to tRNACCA).

    • Challenge: Find the mRNA codon sequence that is complementary to tRNATrp.

Wobble Hypothesis:

  • There are 61 codons coding for amino acids, but fewer than 61 tRNAs.

  • This suggests some tRNAs can read more than one codon, known as wobble.

  • First base of tRNA anticodon sets how many codons it can read:

    • Bases C or A: Can pair with one codon.

    • Bases U or G: Can pair with two codons.

    • Base I: Can pair with three codons.

  • Example: tRNA ext{I} GC (encoding alanine) reads GCU, GCC, and GCA.

Base Pairing Types in Wobble:

  • Canonical A-U and G-C base pairs exist alongside non-canonical pairs:

    • U-G base pair

    • Inosine (I) can pair with A, C, or U.

Preparing tRNAs for Translation:

  • tRNAs must be connected to the correct amino acid before translation.

  • The aminoacyl-tRNA (charged tRNA) features an ester linkage to the 3’ CCA arms of tRNA.

  • The linkage can occur through the 2’ or 3’-OH of ribose.

Steps in Charging tRNAs:

  1. Activation of Amino Acid: Amino acid reacts with ATP to form aminoacyl adenylate, producing pyrophosphate.

  2. Transfer to tRNA: Aminoacyl adenylate (AMP) is then transferred to tRNA, with near-zero free energy of transfer.

  3. Proofreading: Newly-adjacent amino acids are verified after tRNA charging.

Selecting the Right Amino Acid:

  • Aminoacyl-tRNA synthetases must be selective for their amino acids, which is crucial for accurate translation.

  • Example: Addition of threonine must prevent incorrect addition of similar amino acids like valine or serine.

Mechanism of Selecting Amino Acids:

  • Active sites in the synthetases use shape and specific interactions for selection.

  • The double sieve mechanism:

    • Active site excludes oversized AAs.

    • Editing site cleaves undersized AAs that could be incorrectly incorporated.

Synthetase tRNA and Amino Acid Recognition:

  • Recognition involves not only the anticodon loop but also other tRNA regions that might involve modified bases in their structure.

Error Rates and Consequences in Amino Acid Addition:

  • Errors can occur due to incorrect tRNA-amino acid coupling or tRNA-mRNA interactions.

  • Incorrect amino acid frequency per peptide synthesis:

    • At an error rate of 10−510^{-5} to 10−410^{-4}, actual error rates yield a low incidence of functional impairment.

Consequences of Degeneracy:

  • Codon bias can buffer mutations and allow translational control through patterns of synonymous codons.

Altering the Genetic Code:

  • Modification possibilities exist for the genetic code, leading to enhancements such as:

    • Addition of custom amino acids for applications in fluorescence, etc.

    • Creating viral resistance by eliminating synonymous codons.

Ribosomes:

  • Ribosomes utilize charged tRNAs to build proteins.

  • E. coli ribosome structure:

    • 50S Large Subunit: Contains 34 proteins, RNA at 23S and 5S.

    • 30S Small Subunit: Contains 21 proteins in its structure, RNA at 16S.

  • Ribosomes are ribozymes, predominantly composed of rRNA.

Abundance of Ribosomes:

  • E. coli can have up to 100,000 ribosomes per cell.

Co-Transcriptional Translation in E. coli:

  • Translation direction aligns with mRNA production at 5’-to-3’.

  • Access to mRNA facilitates co-transcriptional translation.

Bacterial Transcription Regulation: Attenuation:

  • Trp operon encodes proteins essential for tryptophan biosynthesis.

  • It is regulated by attenuation in response to tryptophan concentrations:

    • High Tryptophan: Ribosomes progress along trp codons forming a stem-loop.

    • Low Tryptophan: Ribosomes stall at trp codons preventing stem-loop formation.

Key Concepts Recap:

Genetic Code Exploration:

  • Important features and functions of the genetic code.

  • The concept of degeneracy and its impact on genetic coding.

  • Commonalities among tRNAs and the procedure for charging.

tRNAs:

  • Question the universality of the genetic code.

Aminoacyl-tRNA Synthetases:

  • Explore potential outcomes from incorrect translations (e.g., mitochondrial proteins).

Understanding Wobble:

  • Investigate which bases facilitate wobble and its implications for accuracy.

Energetics:

  • Examine activation costs for amino acids and the role of pyrophosphate.

  • Investigate how synthetases ensure correct amino acid and tRNA pairing while troubleshooting errors through editing sites.

Co-transcriptional Translation:

  • Grasp the principles of co-transcriptional translation in bacteria.

Translational Coding:

  • Be prepared to utilize the genetic code to translate mRNA sequences effectively.