• Genetic code
• Protein translation
• Ribosome
• Location of Transcription and translation
• Eukaryotic transcription and splicing

How do 64 different codons produce 20 different amino acids?
 

• The start codon is AUG.  Methionine is the only amino acid specified by just one codon, AUG.
• The stop codons are UAA, UAG, and UGA.  They encode no amino acid. The ribosome pauses and falls off the mRNA.
• The stretch of codons between AUG and a stop codon is called an open reading frame (ORF).  Computer analysis of DNA sequence can predict the existence of genes based on ORFs.
• Other amino acids are specified by more than one codon--usually differing at only the third position.

Evolution of the Code
Did codons evolve to correspond to particular amino acids based on chemistry, or did the code evolve at random?
The code evolved at random, in that there is no direct chemical connection between, say, GGG and Glycine.  BUT--the code appears to have evolved along certain lines for logical reasons.  The two most "fundamental" amino acids are Gly and Ala, in biochemical pathways and in natural occurence in prebiotic systems.  Both are specified by G/C pairing at the first two positions--the strongest possible interaction.  Early life, under high-heat conditions, would have needed extra strong codon-anticodon pairing.  The first code may even have been a two-base code. For more evidence and speculation on this topic, see http://www.evolvingcode.net/.

Protein translation

Translation involves the conversion of a four base code (ATCG) into twenty different amino acids.  A codon or triplet of bases specifies a given amino acid.  Most amino acids are specified by more than one codon.

The conversion of codon information into proteins is conducted by transfer RNA.  Each transfer RNA (tRNA) has an anticodon  which can base pair with a codon.  Some anti-codons have modified bases that can pair with more than one codon, specifying the same amino acid;  this means that we don't need 61 different tRNA molecules for all 61 codons. (What do the other three codons specify?)

The structure of transfer RNA (tRNA):

• 3'OH end esterifies with COOH of amino acid:

 This process, called charging, is catalyzed by a tRNA transferase, or aminoacyl tRNA synthetase, specific to the tRNA type.  There are one or more tRNA types, specified by different genes, for EACH amino acid.

• Anticodon loop, capable of complementary base pairing to a codon on the message.  May contain the unusual base inosine, which is capable of binding to more than one base.  The "wobble hypothesis," by Frances Crick in the '60s, first showed how inosine could enable one tRNA to recognize more than one codon.  Otherwise, the cell would need more than 60 different tRNAs.
• Ribosome binding  and tRNA transferase recognition.

Translation of mRNA into polypeptide

Translation requires initiation, elongation, and termination.  Translation is performed by the ribosome, an organelle composed of more than fifty different proteins plus two structural rRNAs, each part of the 30s subunit or the 70s subunit. The "s" is a unit of  sedimentation, referring to how fast a particle settles out during centrifugation.

Note that  this entire process requires tRNAs continually being charged with their respective amino acids, by tRNA transferase enzymes.

(1) Initiation occurs by binding of the 30s subunit to the mRNA.  In bacteria, the mRNA binds by hybridization of a special sequence to the Shine-Dalgarno sequence of the 16s rRNA, part of the 30s subunit.  The ribosome then finds the first AUG sequence on the mRNA, where it binds the anti-codon of a Met-tRNA, at the P site.

(2) Elongation occurs by successive amidation of the nascent (growing) chain.    The 50s subunit now binds, creating the A site. Each new aminoacyl-tRNA enters at the A site, where it transfers the amino end of its amino acid to the carboxylic end of the nascent chain.  The entire ribosome now "translates" over one codon position, so that the nascent chain is now bound to the P site.  Elongation requires energy provided by GTP.

(3) Termination occurs when the A site reaches a stop codon.  Since no tRNA exists with an anticodon complementary to the stop codon, the ribosome "pauses" until at last it "falls off" the mRNA, and the polypeptide chain terminates. This process is facilitated by a release factor protein that binds into the ribosomal A site containing a stop codon to help with protein release.

the Griffiths et al, current edition

Ribosome Model

• In bacteria, nearly all translation occurs on growing mRNA still being transcribed.
• In eukaryotes, 15% of translation occurs on growing mRNA.  The purpose of translating incomplete RNA in the nucleus may be to eliminate errors that result in stop codons terminating the peptide.  The remaining 85% of translation occurs after the mRNA is processed (see below) and exits the nucleus, into the cytoplasm.

If the growing peptide is hydrophobic, to function in the membrane, its hydrophobic signal peptide attaches to the signal recognition particle (SRP).  The SRP is composed of protein and RNA (like the ribosome).  SRP carries the hydrophobic peptide, with its ribosome, to the face of a membrane for insertion:

• In bacteria, the signal peptide is inserted in the plasma membrane.
• In eukaryotes, the signal peptide goes to the endoplasmic reticulum (ER).

Problem: DNA replication, RNA transcription, and protein translation take lots of energy.  Why?
Which processes take more energy than others?

In eukaryotes, production of mRNA is more complicated than in bacteria, because:

• The initial RNA molecule is elongated by one of three different RNA polymerases
• The initial RNA  has to be spliced and processed
• The completed mRNA has to exit the nucleus to be translated in the cytoplasm

Splicing of hnRNA to make mRNA
The first transcript of RNA from a eukaryotic gene is not yet ready for transcription.  It is called hnRNA,  for high-molecular-weight nuclear RNA.  In order for the RNA to exit the nucleus, and for  proteins to be translated by ribosomes in the cytoplasm, the following processing steps must first occur:

• Capping of the 5' sequence with 5' methyl-7-guanidine (the "m-7-G cap")
• Addition of a run of adenine nucleotides to the 3' OH end (the "poly-A tail")
• Splicing out of the intron sequences

The splicing of introns is a complex intramolecular reaction, mediated by an organelle composed of RNA and protein molecules, the spliceosome.The spliceosome catalyzes the reaction between a 2'OH of an Adenine, and the 5' phosphate end of the intron, creating a lariat loop.  (Note: Only RNA has 2'OH to do this!)  The lariat reaction produced a 3'OH on one exon, enabling to join the 5' phosphate of the joining exon.

The spliceosome is actually composed of several small nuclear riboprotein (RNA-protein) organelles, called snRNPs.  To see how these snRNPs (labeled U1, U2 etc.) splice out the intron, view this animation.

Why would cells have evolved to have introns, which seem wasteful of DNA an energy?  Two kinds of reasons have been proposed:

(1)  Organisms first evolved with introns and other pieces of non-coding nucleic acid.  As organisms evolved, the bacteria "lost" the non-coding portions through rapid evolution.  Eukaryotes however evolved more slowly and have not yet "lost" these sequences.

(2)  Non-coding DNA sequence serves functions for the cell.  It may provide  a structural function, a place for chromosome recombination to occur without jeopardizing the integrity of the coding sequences.  It does provide a way for alternate splicing patterns in different tissues to produce slightly different versions of the same gene product.  Thus, RNA splicing actually helps multicellular organisms to economize in their genomes.