Chromosomes consist primarily of proteins and nucleic acid and that fact was known in the late 1800’s, but what wasn’t known was how they operate, and which type of molecule (the protein or the nucleic acid) was responsible for heredity

Several key experiments led up to the modern era of molecular biology:

1920’s – Frederick Griffith – studying two strains of Streptococcus pneumoniae, he showed that one strain could be ‘transformed’ to produce a capsule that is necessary for the bacterium to cause pneumonia. What was the transforming factor?

1944 – Avery, MacLeod and McCarty – repeated Griffith’s experiments with some modifications. They isolated chromatin from the bacteria, then treated it with either a protease (breaks down protein) or a nuclease (breaks down DNA). After treating the chromatin with one of the enzymes, they ‘transformed’ the chromatin into the naked bacteria. Chromatin treated with the protease could still transform the bacterial cells, but chromatin treated with the nuclease could not transform. This indicated that DNA was responsible for passing on genetic information.

1952 – Hershey and Chase showed definitively that DNA was the genetic material. Their experiment involved radioactively labeling a bacerial virus (bacteriophage T2), allowing the phage to replicate in host cells then determining where the radioactivity ended up. The two radioactive labels they used were Sulfur35 which would only be found in proteins and Phosphorus32 which is found in nucleic acid. Only the offspring of viruses labeled with P32 retained any radioactivity so they concluded that DNA was the genetic information molecule that is passed on through cell division.

1953 – J.D. Watson, Francis Crick, and Maurice Wilkins determine the structure of DNA. They were awarded the Nobel prize in 1962. The sugar deoxyribose and phosphates make the sides of the double helix. The nitrogen bases form the rungs of the ladder. There are four nitrogen bases in DNA: two purines (adenine and guanine) and two pyrimidines (thymind and cytosine). "A" always forms a base pair with "T" and"G" always pairs with "C". These are called complementary base pairs. Each strand of DNA is complementary base paired with the neighboring strand, but the direction of the two strands is oriented differently with respect to the phosphate bonds connecting the nucleotides. This makes DNA antiparallel.

DNA Replication – occurs using a semi-conservative method (Meselson and Stahl).

1. Unwinding parental strands – this stage involves the enzyme DNA helicase, which unwinds and separates two strands of DNA.

2. Synthesis of complementary base pairs – DNA polymerase works only in one direction to add complementary nucleotides. The parent strand serves as a template. DNA polymerase also has a proofreading function. It can ‘swap’ mismatched nucleotides.

3. Okazaki fragments. While DNA polymerase synthesizes one strand continuously, the second strand is made discontinuously in short fragments. (This is due to the antiparallel nature of DNA; the polymerase reads the template from 3’ to 5’, and the new strand is synthesized from 5’ to 3’). The short fragments are linked together by DNA ligase.

Transcription – uses DNA as a template to synthesize RNA.

Differences between DNA and RNA

1. RNA is a single strand

2. Ribose is the sugar

3. Uracil replaces Thymine as a nitrogen base

DNA from selected genes is copied into RNA. Only one strand of the DNA is transcribed. (Remember: DNA strands are complementary, not identical).

3’ G A C G T A A C T 5’ DNA antisense strand

G U C G U A A C T RNA

5’ C T G C A T T G A 3’ DNA sense strand

Transcription takes place in three stages:

Initiation. The enzyme RNA polymerase adds ribonucleotides to a growing strand. RNA polymerase must be able to locate the beginning of a gene. A promoter is a short sequence of DNa located 3’ (before) the gene’s beginning. RNA polymerase binds to this region of the DNA.

Elongation. RNA polymerase separates the 2 DNA strands and begins adding new bases to the growing strand.

Termination. RNA polymerase usually ‘runs’ past the end of a gene, until it reaches a DNA sequence that ‘tells’ the RNa polymerase to stop and the RNA is released. RNA polymerase and the DNA template separate.

Transcription can make 3 types of RNA.

Messenger RNA: mRNA carries the genetic code for proteins to ribosomes. (In eukaryotic cells, mRNA travels through pores in the nuclear envelope to the cytoplasm.)

Ribosomal RNA: Once rRNA has been transcribed, it becomes a part of the ribosomes in the cytoplasm. rRNA is thought to have enzymatic activity involved in adding amino acids to a protein chain.

Transfer RNA. tRNA’s bind to amino acids and deliver them to ribosomes where the amino acid is ‘transferred’ to the new protein chain.

The genetic code carried in the mRNA must be translated by the ribosomes. It takes 3 nucleotides to code for one amino acid. The triplets of 3 nucleotides are called codons. The code is said to be degenerate because more than one codon may specify the same amino acid. For example:

UCU, UCC, UCA, UCG All four of these codons signify the amino acid serine. The first two nucleotides are the same and only the 3^rd position varies. The 3^rd position is sometimes called the ‘wobble’ position.

Three codons do not correlate to any amino acid and they are called "stop" codons. There is at least one stop codon at the end of every mRNA. Most mRNA’s also have a "start" codon, AUG. This is the amino acid methionine, and most proteins have that as their first amino acid.

Mutations are permanent changes in DNA sequence that may have a drastic effect on the proteins for which they code.

Point mutations

Deletions

Insertions

The same terms, initiation, elongation, and termination are used to describe the stages of translation.

Initiation – involves the assembly of the mRNA and ribosome complex. The first codon that the ribosome reads is an AUG, or methionine. a tRNA carrying methionine brings the amino acid to the ribosome and initiates protein synthesis.

Elongation – There are 2 ‘docking’ sites in the ribosome, the A site and the P site. The P site is where the protein chain is located and the A site is where the tRNA brings in the appropriate amino acid. tRNA binds to the A site and the growing polypeptide moves over to the A site, picking up the new amino acid. Then it promptly moves back to the P site and the previous tRNA is released so that a new tRNA can bring in the next amino acid.

Termination – When the ribosome reaches a stop codon, the polypeptide is released and the ribosomal subunits disassociate.

The structure of tRNA is critical in translation. At one end of the molecule is the amino acid binding site. At the opposite end is an ‘anticodon’ or bases complementary to the mRNA’s codon. This is how the tRNA can ‘dock’ or bind to the mRNA, bringing the right amino acid into proximity with the growing polypeptide chain.