Lecture 25: The Genetic Code

General characteristics of tRNA molecules include the following:

1. Each is a single strand of RNA between 73 and 93 ribonucleotides in length. 2. The three-dimensional structure of the molecule is L-shaped. 3. Transfer RNA molecules contain unusual bases such as inosine or bases that have been modified. 4. In a two-dimensional representation, all tRNA molecules appear as a cloverleaf pattern. The amino acid-accepting region is the acceptor stem, which contains the 3' CCA terminal region. Many of the nucleotides are involved in hydrogen bonds that form stems and loops. 5. The 5' end is phosphorylated, and the 5' terminal residue is usually pG. 6. The amino acid is attached to a hydroxyl group of adenosine in the CCA region of the acceptor stem. 7. The anticodon is in a loop near the center of the sequence.

Wobble

is tolerated in the third position of the codon because, while ribosomal RNA monitors fidelity of the base-pairing in the first two positions of the codon-anticodon complex, ribosomal RNA does not evaluate the correctness of the third position.

In order to be incorporated into proteins, amino acids must be activated.

Amino acids are activated by formation of an ester linkage between the carboxyl group of the amino acid and either the 2' or 3' hydroxyl group of the terminal adenosine of the tRNA, forming an aminoacyl-tRNA or charged tRNA. Aminoacyl-tRNA synthetases catalyze the activation of amino acids. The first step is the formation of aminoacyl adenylate The aminoacyl group is then transferred to a specific tRNA recognized by the synthetase The aminoacyl-AMP never leaves the active site of the synthetase.

Amino Acids are Activated by Attachment to Transfer RNA

Aminoacyl-tRNA synthetasescatalyze the activation of amino acids by forming an ester linkage between the carboxyl group of the amino acid and either the 2' or 3' hydroxyl group of the terminal adenosine of the tRNA, forming an aminoacyl-tRNA or charged tRNA

There are 5 key characteristics of the genetic code

Characteristics of the genetic code 1. Three nucleotides, called a codon, encode an amino acid. 2. The code is nonoverlapping. 3. The code has no punctuation. 4. The code is read in the 5'-to-3' direction. 5. The code is degenerate in that some amino acids are encoded by more than one codon.

Aminoacyl-tRNA Synthesaes Have Highly Discriminaating Amino Acid Activation Sites

Each aminoacyl-tRNA synthetase is specific for a particular amino acid. Specificity is attained by various means in different enzymes

The Synthesis of Long Proteins Requires a Low Error Frequency

Error frequencies of 10^-4 slow for the accurate synthesis of even large proteins at a very rapid rate

The ribosome is the site of protein synthesis. In E. coli, the ribosome sediments at 70S and is composed of two subunits, a large 50S subunit and a smaller 30S subunit.

The 50S subunit is composed of 34 proteins and a 23S RNA as well as a 5S RNA. The 30S subunit contains 21 proteins and a molecule of 16S RNA.

Ribosomes have 3 tRNA-binding sites that bridge the 30S & 50S subunits

The A (aminoacyl) site binds the incoming tRNA. The P (peptide) site binds the tRNA with the growing peptide chain. The E (exit) site binds the uncharged tRNA before it leaves the ribosome.

The Genetic Code Is Nearly Universal

Most organisms use the same genetic code. However, some organisms ad organelles have slight modifications Recent research revealed that different organisms may prefer different sets of synonymous codons, although the biochemical benefit of codon bias is not yet clearly established. Most organisms use the same genetic code. However, some organisms have slight modifications. For instance, in ciliated protozoa, codons that are stop signals in most organisms encode amino acids. Mitochondria also use variations of the genetic code.

Amino acids are encoded by groups of three bases starting from a fixed point

Open reading frames (ORFs) start at the start codon (AUG), which codes for methionine, and end at one of three stop codons

Mutations in DNA can cause changes in protein sequence

Point mutations can be silent, nonsense or missense mutations. Insertions or deletions cause frameshift mutations

Each aminoacyl-tRNA synthetase is specific for a particular amino acid.

Specificity is attained by various means in different enzymes. Threonyl-tRNA synthetase contains a zinc ion at the active site that interacts with the hydroxyl group of threonine. Valine is similar in overall structure to threonine but lacks the hydroxyl group and thus is not joined to the tRNAThr. Serine, although smaller than threonine, is occasionally linked to tRNAThr because of the presence of the hydroxyl group.

The Genetic Code Links Nucleic Acid and Protein Information

Protein synthesis is a process of translation. Nucleic acid sequence information is translated into amino acid sequence information. The genetic code links these two types of information

Wobble: What is it

Some tRNA molecules can recognize more than one codon. The recognition of the third base in the codon by the anticodon is sometimes less discriminating, a phenomenon called this

The anticodon forms base pairs with the codon

Some tRNA molecules can recognize more than one codon. There recognition of the third base in the codon by the anticodon is sometimes less discriminating, a phenomenon called wobble

A ribosome is a Ribonucleoprotein Particle Made of Two Subunits

The ribosome is the site of protein synthesis. In E. coli, the ribosome sediments at 70S and is composed of two subunits, a large 50S subunit and a smaller 30S subunits

The general structure of transfer RNA molecules.

The structure of the tRNA molecule is shown in the cloverleaf pattern. Comparison of the base sequences of many tRNAsreveals a number of conserved features.

By convention, sequences are written in the 5'-to-3' direction

Thus, the anticodon that pairs with AUG is written as CAU.

Transfer RNA Molecules Have a Common Design

Transfer RNA (tRNA) molecules function as an adaptor molecule between a codon and an amino acid. There is at least one tRNA molecule for each amino acid. There are 7 key characteristics

Ribosomal RNAs Play a Central Role in Protein Synthesis

Two-Thirds of the mass of ribosomes in RNA, which is critical for the structure and function of the ribosome. The ribosomal RNAs fold into complex structures with many short duplex regions. Ribosomal rNA is the actual catalyst for protein synthesis, with the ribosomal proteins making only a minor contribution

A mutated version of the gene to be disrupted is constructed, maintaining some regions of homology with the normal gene (red)

When the foreign mutated gene is introduced into an embryonic stem cell, recombination takes place at regions of homology The normal (targeted) gene is replaced, or "knocked out", by the foreign gene. The cell can then be inserted into embryos, and mice lacking the gene (knockout mice) are produced.