Lecture 4

 
 

Polypeptide Synthesis Overview

 
 
Polypeptide synthesis proceeds sequentially
from N Terminus to C terminus.
 
 
Amino acids are not pre-positioned on a template.  Proved by the classic experiment of Dintzis in 1961.  He synthesized hemoglobin in a test tube using a reticulocyte (red blood cell) system.  He initiated protein synthesis and then added H3-labeled leucine.  He isolated hemoglobin, partially labeled with incorporated H3-leucine, and created peptide fragments by digestion of hemoglobin with trypsin.  Then he analyzed peptide fragments to determine the relative amount of radioactivity.   He obtained the following results:
 
 
 
 

Figure 4.1.1: Incorporation of radioactive leucine into hemoglobin after different periods of incubation.  There is a gradient from the C-terminus to the N-terminus of the protein, which implies that synthesis takes place sequentially from N-terminus to C-terminus.  As the time of incubation is increased, the total amount of radioactivity is increased but the gradient remains.  Results are expressed as the ratio of an H3 label (tritium) in leucine to an internal control of C14.  Data of Dintzis (1961).
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Ribosomes read mRNA in the 5'->3' direction,
not the 3'->5' direction. 
 
To prove, one must know the Genetic Code.  For example, AAA codes for Lys, AAC codes for Asn and CAA codes for Gln.  In a cell-free protein synthesizing system, initiate protein synthesis by adding artificial mRNA, consisting of polyA with one C (cytosine) at the 3' end.  Analyze the N and C termini of the peptide fragments.
 
 
Nucleotide Sequence
Protein Sequence
Direction of Synthesis
5'-AAAAAA(AAA)nAAC-3'
Lys-Lys-(Lys)n-Asn
5' -> 3'
3'-CAAAAA(AAA)nAAA-5'
Gln-Lys-(Lys)n-Lys
3' -> 5'
 
 

Next:  Polypeptide Synthesis Overview (continued)

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