Genetic Code

Brief Overview of Protein Synthesis (Translation)

Figure 5.20 gives a brief, but somewhat inaccurate, picture of translation (protein synthesis).

A few important facts:

• mRNA is translated 5' to 3' (remember that RNA is synthesized in the 5' to 3' direction).
• Proteins are made in the direction amino terminus to carboxyl terminus.
• Peptide bonds are synthesized at a rate of about 15 amino acids per second.
• Since three nucleotides constitute a codon for an amino acid, this means that the ribosome "moves" along the messenger RNA at the rate of about 3 x 15 = 45 nucleotides per second; that is the rate of translation is just as fast as the rate of transcription.
• In fact, since E. coli does not have a nucleus, transcription and translation are coupled together (even before a messenger RNA is finished being made, protein synthesis can begin on it).
• Many ribosomes translate a messenger RNA at the same time (this is known as a polysome, or polyribosome).

Figure 27.5 shows the organization of the lac operon messenger RNA (a polycistronic mRNA). It shows the open reading frames for lac Z, lac Y, and lac A genes as well as the start codons and stop codons for each gene. There are untranslated regions on the lac messenger: leader, trailer, and intergenic regions between each gene. The start codons establish a reading frame for each gene.

The Code Problem

There are only four nucleotides in RNA but there are twenty amino acids in proteins (Figure AH).

The Genetic Code (Figure 27.1) is, in fact, non-overlapping, punctuation-free (except for start and stop signals), and degenerate (but NOT ambiguous) where each amino acid is specified by at least one triplet nucleotide combination (codon). The Genetic Code is universal; that is, virtually all organisms use the same code (there are a few minor exceptions, but they are rare).

Colinearity of Gene and Polypeptide

Charles Yanofsky mapped mutations in the E. coli tryptophan synthetase A (trpA) gene. He also determined the amino acid sequence of the wildtype protein as well as the sequences of each mutant protein. This showed that genes are colinear with polypeptide chains (Figure AI). This work also showed that the Genetic Code is NOT an overlapping code since one mutation only changes one amino acid in the protein (not three amino acids!). Mutations at two different sites in the trpA gene affect the same amino acid (number 211 shown circled below) in the tryptophan synthase alpha subunit; this was the first evidence that more than one nucleotide specified a given amino acid!

Frameshift Mutations

Mutagens like proflavin and acridine orange are flat, aromatic molecules about the size of a base pair. These chemicals cause frameshift mutations (insertions or deletions of base pairs in DNA). Figure AJ shows genetic experiments done with Phage T4 gene e (the gene coding for lysozyme) frameshift mutants.

• Insertions (+ mutants) or deletions (- mutants) shift the reading frame (frameshift mutations).
• Recombinants between + and - mutants are often phenotypically wildtype (pseudo wildtype) since an insertion together with a deletion in the gene (cistron) restores the reading frame (intragenic supression). Of course, the amino acids in the region between the + and - mutations are still "scrambled", so the pseudo wildtype protein must be at least partially functional with these changes.
• Two + mutations or two - mutations clustered in the same gene are still mutant.
• Three + mutations or three - mutations clustered in the same gene are often pseudo wildtype.
• Thus, the Genetic Code is at least a multiple of three nucleotides (and most likely just three nucleotides).
• Therefore, the code is degenerate.
• The code is read sequentially from a fixed point.
• The code has no internal punctuation (that is, no internal signals for "spaces" or "commas", etc.).

Translation of Synthetic Messenger RNAs

Synthetic messenger RNAs could be translated in biochemical reactions that catalyzed protein synthesis (Figure 27.2). These experiments are described on page 1004. (Although all twenty amino acids are present in these reactions, only one amino acid is radioactively labeled). These synthetic mRNAs did not have a specific start site for translation, so protein synthesis started randomly on any given mRNA (that is, there was random choice of one of the three possible reading frames).

In the complicated synthetic mRNA in this figure, the possible codons are AAG, AGA, and GAA. Although only three polypeptides are synthesized under its direction (polylysine, polyarginine, and polyglutamate), this is still not enough information to unambiguously assign codons.

Translation of poly U gave only polyphenylalanine (UUU = phe). Translation of poly CCC gave only polyproline (CCC = pro). Translation of poly A gave only polylysine (AAA = lys).

Already we can see that at least TWO codons must code for lysine: AAA and either AAG, AGA, or GAA.

Filter Binding Assay

Figure AK shows experiments described on pages 1004-1005.

Marshall Nirenberg and his collaborators used extracts of E. coli plus chemically synthesized trinucleotides to decipher the entire genetic code. Twenty mixtures of aminoacyl-tRNAs (tRNAs that have an amino acid attached) were prepared. In each mixture, a different amino acid was radiolabeled (color); the other 19 amino acids were present on tRNAs but they were unlabeled. An aminoacyl-tRNA sample and a trinucleotide would pass through a nitrocellulose filter without binding (left panel). Ribosomes, however, would bind to the filter (center panel). When samples from each of the 20 aminoacyl-tRNA mixtures were mixed with ribosomes and a trinucleotide and then filtered, one of the 20 samples would leave a labeled complex stuck to the filter. In the example shown here, the complex consists of the ribosome, the trinucleotide UUU, and the labeled phenylalanyl-tRNA. This indicates that UUU is recognized by the tRNA for phenylalanine (but not, for example, by the tRNA for leucine or arginine; when these amino acids were labeled, no labeled complex stuck to the filter with the trinucleotide UUU). Because all possible trinucleotides could be synthesized and tested, this experiment made an enormous contribution to the work on the genetic code. (Science 145: 1399, 1964)

The amino acids specified by about 50 codons were quickly identified in this way. For the remaining codons, the filter binding assay was either negative (no tRNA bound) or uncertain. However, all of the codons identified in the filter binding assay agreed with the much fewer codons identified using synthetic mRNAs in biochemical protein synthesizing reactions.

The Genetic Code

	U	C	A	G
U	Phenylalanine	Serine	Tyrosine	Cysteine	U
					C
	Leucine		STOP	STOP	A
				Tryptophan	G
C	Leucine	Proline	Histidine	Arginine	U
					C
			Glutamine		A
					G
A	Isoleucine	Threonine	Asparagine	Serine	U
					C
			Lysine	Arginine	A
	Methionine				G
G	Valine	Alanine	Aspartic Acid	Glycine	U
					C
			Glutamic Acid		A
					G

The important codons to remember are the start codon (5'-AUG-3' specifying methionine) and the three stop codons (5'-UAA-3', 5'-UAG-3', and 5'-UGA-3'). All proteins start with the amino acid methionine (Note: Formylmethionine, the actual initiating form of methionine, is the first amino acid at the amino-terminus of the protein. Internal methionines (not formylated) are also coded for by AUG. Formylmethionine is also the first amino acid even when GUG or UUG (the lacA gene uses this rare start codon!) are used as initiation codons.). The start codon sets up a reading frame: all subsequent codons are read in order until a stop codon terminates protein synthesis.

Types of Mutations

Figure AM shows a missense and a nonsense mutation. Other useful terms are: silent mutation, silent sites, blocked reading frame, closed reading frame, neutral substitutions, nonsense codon, , ochre codon, ochre mutation, open reading frame, point mutation, stop codons, amber codon, amber mutantion.