THE GENETIC CODE, pp. 97-99.

 

A. The GENETIC CODE involves a mechanism by which the specific nucleotide sequence found in a messenger RNA is exactly converted to the primary amino acid sequence of the corresponding protein. Fig. 3.13, 3.14 and Table 3.1.

B. Remember that there are only 4 possible nucleotides in RNA, but there are 20 possible amino acids in proteins. Therefore the nucleotide sequence is read as groups of 3 adjacent bases at a time called CODONS. There are 43 = 64 possible codons which implies that there exist more than one codon per amino acid. This is called DEGENERACY.

C. Mutations result from changes to the gene’s DNA sequence. These changes may have different effects on the gene’s protein sequence, as a result of the genetic code. For example, point mutations are those that involve single base pair changes:

1. Changing one nucleotide in a codon may lead to a:

Missense mutation: changes one amino acid to another.

Nonsense mutation: changes an amino acid codon to a stop codon

Silent mutation: no change in the protein sequence results.

2. An insertion or deletion of one base pair leads to a Frameshift mutation. The effects of a frameshift mutation can sometimes be at least partially alleviated by a second frameshift mutation downstream that restores the proper reading frame.

How Do We Know?

1. The Genetic Code? The Code was determined in the early 60's when few protein sequences were known and before any mRNAs were purified or any DNA sequences determined. Matthei and Nirenberg began the process by developing cell free protein synthesis systems (basically a complex cytoplasmic soup containing ribosomes and everything else needed). They then used the enzyme Ochoa had discovered to make artificial RNAs such as polyuridylic acid (UUUUUUUUU--). When they added this to their extracts and tested to see which radioactively-labeled amino acid was made into polypeptides, they found that only phenylalanine worked, so they said UUU=Phe. (Of course, polyuridylic acid doesn't contain an AUG, but these in vitro systems will occasionally initiate almost anywhere.) Similarly, the amino acids for CCC, GGG, and AAA could be determined, but the other codons remained unknown. Then P. Leder and Nirenberg showed that ribosomes when provided with a three nucleotide long RNA (a "triplet", these were small enough that they could easily be synthesized by organic chemistry then) would often bind only one specific labeled aminoacyl-tRNA, e.g., if you gave them UUU, they would bind only Phe-tRNA; if you gave them CUU, they would bind Leu-tRNA. This helped derive several other codon identities, but didn't work in all cases. H.G. Khorana was developing organic chemical techniques to synthesize DNA (we now have machines that do this based mostly on the techniques Khorana and his students perfected). At the time, he still could only make small DNAs on the order of 6-12 bases in each strand, but these were big enough that RNA polymerase would use them to make specific RNAs to test in the protein synthesis extracts. For example, he could make CACACACACACA-- and show that it led to the synthesis of HisThrHisThrHisThr--, so CAC is either His or Thr and ACA is the other of the two. Then he could make CAACAACAACAACAACAA-- and show that it made poly(Gln), poly(Asn) and poly(Thr), so ACA=Thr and CAC=His; CAA=Gln or Asn and AAC=Asn or Gln. Eventually, all the code could be determined in this fashion, as later proven by sequence analysis of complex proteins, and DNAs or RNAs. Note that the ability to make a functional protein synthesis cell extract is critical to all of this because intact cells will not take up adequate amounts of RNAs through their plasma membranes to do the assay.

 

TRANSLATION, pp.273-289. Protein synthesis (translation) is a complex, highly regulated process. We will focus on what the ribosome and the tRNAs are doing. In both the initiation and elongation stages, there are a number of other translation factors involved (Table 7.1) which regulate these processes and couple them to the hydrolysis of GTP. You will not need to know the names of these factors.

 

A tRNAs, tRNA synthetases and tRNA CHARGING, (Fig. 3.12, 7.1, 7.2, 7.3.) Note that codons have very different chemical structures than amino acids. Therefore, cells require an ADAPTOR SYSTEM, which uses tRNAs + tRNA SYNTHETASES.

Each amino acid has one or more SPECIFIC tRNA SYNTHETASE which catalyzes the following 2 step reaction (Fig. 7.2):

1. A.A.X + ATPÞ AMINOACYL-ADENYLATEX + PYROPHOSPHATE (PPi)

2. AMINOACYL-ADENYLATEX +tRNAXÞ AMP + AMINOACYL-tRNAX (CHARGED)

B. INITIATION, Fig. 7.8-7.10:

1. The SMALL RIBOSOMAL SUBUNIT binds mRNA at the initiation AUG codon (Fig. 7.7) and the INITIATOR tRNA (methionine or formylmethionine)

2. The LARGE RIBOSOMAL SUBUNIT binds to complete the complex

 

C. ELONGATION, Fig. 7.11:

1. The appropriate next charged tRNA binds to the A SITE.

2. PEPTIDYL TRANSFERASE ACTIVITY forms a peptide bond.

3. In TRANSLOCATION the ribosome moves down mRNA by 1 codon moving the peptidyl tRNA from the A SITE to the P SITE

4. Steps 1. to 3. repeat in order until a stop codon enters the A SITE.

 

D. TERMINATION, Fig. 7.13

1. A STOP CODON in the A SITE triggers hydrolysis of the peptide-tRNA BOND (in the P SITE) and the release of the completed protein and free tRNA.

 

DNA, RNA, AND PROTEIN SYNTHESIS IS ALWAYS DIRECTIONAL:

A. New DNA is always made in the 5' to 3' direction. Therefore the template DNA strand is being copied from 3' TO 5' (i.e., from its 3' end towards its 5' end) since the two strands must be antiparallel.

B. RNA is always made in the 5' to 3' direction. Again the template DNA strand must be read from 3' TO 5'.

C. Protein is made by reading mRNA in the 5' to 3' direction and making new protein from its amino end (N-terminus) towards its carboxyl end (C-terminus). (Fig. 7.8) Several ribosomes read a single mRNA at once, forming a polysome (Fig. 7.14)

(Exercise: Draw a picture of a prokaryotic DNA replication complex and RNA polymerase acting at two different spots on the same DNA molecule. Also draw one or more ribosomes making protein off of the end of the new RNA being transcribed. Show the 5' to 3' direction of each strand of nucleic acid and the N to C terminus direction of the protein.)

How Do We Know?

1. That protein is synthesized N-terminus to C-terminus. In 1963 H. Dintzis pulse-labeled red blood cells with radioactive amino acids for various times and then purified full length a -globin protein and fragmented it into small peptides. By this time V. Ingraham had determined enough of the amino acid sequence of this protein to know the order of each peptide from N-terminus to C-terminus. Dintzis showed that pulse label first showed up in full length protein in the C-terminal peptide meaning it must be synthesized last. As pulses got longer label began to appear in peptides closer and closer to the N-terminus.

2. That ribosomal RNA participates in the catalysis of peptide bond formation? (pp. 280-281)

Food for thought questions

1. The building of polymers like DNA, RNA and protein is an endergonic process (i.e., positive D G° ). From what source does the cell derive the energy to build these polymers in DNA replication, RNA transcription, and protein synthesis?

2. The genetic code is almost universal ; that is, it is exactly the same in virtually all living organisms (there are a few differences used only in the mitochondrial DNAs). Why do you think it is nearly universal?

3. Can you think of a way in which a silent mutation in a codon could still have a deleterious effect (phenotype) on the mutant organism?