• One Gene, One Enzyme
• RNA Transcription
• Mechanism of Transcription
• Transcription and Translation
• Genetic Code

Genes specify proteins. A defective protein leads to an inherited disorder such as PKU.

Using genetic and biochemical approaches with bread mold (Neurospora crassa), Beadle and Tatum devised the first hypothesis about the functional nature of genes.

The first step for expression of any gene function is transcription of RNA.

A famous investigator of the mechanisms of transcription and translation of genetic information is Kenyon alumnus Harvey Lodish. For information on his research on gene expression in blood cell development:Lodish Research.
An example of the role of transcription and translation is regulation of Erythropoietin, the red blood cell-increasing hormone.

A drop in the pressure of oxygen in the blood, such as is caused by ascension to a high altitude, or a major bleeding episode, or anemia, causes certain cells in the kidney to transcribe and translate erythropoietin (Epo) and release it into the blood. Epo activates transcription of other genes in blood cell precursors to develop into red cells, thus increasing the oxygen carrying capacity of the blood. When the oxygen pressure in the blood returns to normal, Epo production returns to its low basal levels. 

But some athletes use artificial injection of Epo to increase their blood cell count to dangerously high levels.

 Ribonucleic acid (RNA) is like DNA except:

• The 2' carbon of the sugar, ribose, has a hydroxyl (OH).  This sugar form is more "fundamental" in the biochemical pathway; deoxyribose (for DNA) has to be synthesized by removal of the 2' OH.  For this reason, we believe RNA evolved first as the genetic material of cells.
• The base uracil replaces thymine.  Uracil lacks the methyl group of thymine.  Biochemically, thymine is made by methylating uracil.
• Most RNA in cells is single-stranded.  Some RNAs, such as tRNA, make complex intramolecular base pairs, and have modified unusual bases.






from T.M. Terry, U Connecticut

Some RNA molecules themselves are the final functional product of the gene from which they are transcribed.  These include:

• Transfer RNA (tRNA).  tRNA molecules attach to specific amino acids and match them to the correct codon during protein synthesis (see below).
• Ribosomal RNA (rRNA).  Each subunit of the ribosome (see below) has an rRNA molecule which holds together the protein components of the ribosome structure.
• Small RNA molecules with enzyme activity (sRNA).  Many other classes of functional RNAs participate as enzyme subunits, as splicing components, and as regulator of transcription.   These are believed to be ancient remnants of the "RNA World" before DNA-based cell evolved. 
• Antisense RNA.  In bacteria, some RNA molecules are transcribed as the complement of genes whose expression can be turned off by antisense RNA hybridization to the messenger RNA.

All classes of RNA molecule need to be transcribed, or copied, from the DNA gene.
This process involves:

• Initiation.  The initiation complex requires:

(1) Promoter sequencewhere RNA polymerase (RNA Pol) recognizes where a gene starts.
(2) Sigma factor joins core RNA polylmerase to bind promoter (in bacteria).  Eukaryotes use transcription factors instead.
(3) Melting of the DNA helix so that the RNA polymerase can bind to exposed bases.
 
• Elongation.  For RNA Pol to get past the promoter:

(1) Sigma factor (or transcription factors, in eukaryote) comes off.
(2) RNA Pol now binds non-specifically, and loosely, to the DNA; it  glides through the helix, while it sequentially adds rNTPs.   Removal of pyrophosphate (PPi) provides energy.
(3) The rNTPs have to be complementary to the template strand of DNA.  The coding strand of DNA (same sequence as RNA transcript) does not actually participate in the reaction.
 
• Termination.  Transcription terminates at short DNA sequences recognized as termination signals.  Some genes require additional termination proteins.

Molecular view

The details of regulation of transcription--how the molecules "know" where a gene begins and ends, and when and where to express it--are very complex and interesting.  We will get into this more after Spring Break.
 

Transcription and Translation

In prokaryotes and eukaryotes:
The RNA encoding proteins starts getting translated by the ribosome, even before the RNA strand is completed. Translation produces a peptide of linked amino acids, which is completed to form a protein.

Note: In high school you probably learned that translation by ribosomes does "not" happen in the nucleus.  Recent research, however, shows that 15% of translation occurs by ribosomes in the nucleus!  The other 85% occurs in the cytoplasm, after the mRNA leaves the nucleus.

In eukaryotes only:
In the nucleus (where some translation occurs), the mRNA encoding proteins gets spliced to cut out introns, then processed (chemically modified at each end).  After splicing and processing the mRNAe leaves the nucleus through nuclear pores in the nuclear membrane.

How do 64 different codons produce 20 different amino acids?
 

• The start codon is AUG. Methionine is 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 (except for Trp and Met as noted above)are specified by more than one codon--usually differing at only the third position. 

Evolution of the Code
Although variations exist in some organisms, the standard genetic code is evolutionarily ancient. 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, http://www.evolvingcode.net/