Genetic Information Experiments: Avery to RNA

Griffith's Transformation Experiments

• Griffith proposed that a transforming principle, later identified as DNA, was necessary for capsule synthesis in bacteria.
• He observed that heat-killed virulent bacteria could transfer their virulence to live non-virulent bacteria.

Avery, MacLeod, and McCarty's Experiments

• They identified DNA as the transforming principle.
• They demonstrated that DNA, not protein, was responsible for transferring genetic information.

Hershey and Chase Experiment

• They used radioactive isotopes to label DNA (³²P) and proteins (³⁵S) in bacteriophages.
• They determined that DNA, not protein, entered the bacterial cells during phage infection.

Hershey and Chase Conclusion

• Phage DNA, not protein, contains the genetic information required for the production of new phages.

Role of RNA in Certain Viruses

• Some viruses use RNA instead of DNA as their genetic material.
• These viruses can directly use their RNA to produce proteins, bypassing the need for DNA.

Evidence Used by Hershey and Chase

• The ³²P labeled DNA was found inside the bacterial cells, while the ³⁵S labeled protein remained outside.

Central Part of Viruses

• Some viruses contain a central core of DNA, while others contain RNA.

Tobacco Mosaic Virus

• The RNA of the tobacco mosaic virus acts as the genetic material.
• Its function is revealed by its ability to direct the synthesis of viral proteins.

DNA Structure

• Watson and Crick proposed the double helix structure of DNA.
• It consists of two antiparallel strands of nucleotides linked by hydrogen bonds.

Antiparallel Chains

• The two chains of DNA run in opposite directions (5' to 3' and 3' to 5').
• This arrangement allows for proper base pairing.

Base Pairing

• Adenine (A) pairs with thymine (T) via two hydrogen bonds.
• Guanine (G) pairs with cytosine (C) via three hydrogen bonds.

Spacing Between Bases

• The nitrogenous bases in DNA are spaced 0.34 nm apart.

RNA Sugar

• RNA contains ribose sugar, while DNA contains deoxyribose sugar.

RNA Involved in Protein Synthesis

• Messenger RNA (mRNA) carries genetic information from DNA to ribosomes.
• Transfer RNA (tRNA) delivers amino acids to the ribosomes for protein synthesis.
• Ribosomal RNA (rRNA) combines with proteins to form ribosomes.

Role of snRNAs

• They participate in the processing of mRNA, including splicing and capping.

Electrical Charge of Nucleic Acids

• Nucleic acids have a negative charge due to the phosphate groups in their backbone.

UV Light Absorption

• Nucleic acids absorb UV light most effectively at 260 nm.

Stable Base Pairing

• Guanine (G) and Cytosine (C) pairing is more stable due to three hydrogen bonds.

In Vitro DNA Synthesis

• Kornberg's experiments revealed the requirements for in vitro DNA synthesis:
• DNA polymerase, a template DNA strand, deoxynucleoside triphosphates (dNTPs), and magnesium ions (Mg²⁺).

Direction of DNA Synthesis

• DNA synthesis occurs in the 5' to 3' direction.
• New nucleotides are added to the 3' end of the growing strand.

Denaturation

• During denaturation, the hydrogen bonds between DNA strands break.
• The double helix separates into two single strands.

Renaturation

• Renaturation allows for the reassociation of complementary DNA strands after denaturation.

Energy for DNA Synthesis

• The energy for nucleotide addition comes from the hydrolysis of the high-energy phosphate bonds in dNTPs.

Semi-Conservative Replication

• Each new DNA molecule contains one original strand and one newly synthesized strand.

Meselson-Stahl Experiment

• It confirmed the semi-conservative mode of DNA replication.
• They showed that after one round of replication, DNA molecules have an intermediate density.

Density of Newly Synthesized DNA

• When E.coli is transferred from a medium with ¹⁵N to one with ¹⁴N, the density of newly synthesized DNA decreases.

E. coli Origins of Replication

• E.coli has one origin of replication (oriC).

Initiation of Prokaryotic DNA Duplication

• The origin of replication (oriC) is recognized by specific proteins that initiate DNA duplication.

Strand Separation

• The two strands of DNA separate during the initiation stage of replication.

Bacterial Replication

• Bacterial DNA replication is bidirectional, meaning it proceeds in both directions from the origin of replication.

Conservative Replication

• In conservative replication, the original DNA strands remain together, and two completely new strands are synthesized.

Single-Stranded Binding Protein

• It binds to single-stranded DNA during replication, preventing them from re-annealing.

Topoisomerase

• It relieves the tension ahead of the replication fork by cutting and rejoining DNA strands.

DNA Pol I

• It removes RNA primers and replaces them with DNA nucleotides.
• It also has a 3' to 5' exonuclease activity for proofreading.

DNA Pol III

• It is the primary enzyme responsible for synthesizing new DNA strands.
• It has a 5' to 3' polymerase activity and a 3' to 5' exonuclease activity.

Primase

• It synthesizes short RNA primers that provide a starting point for DNA polymerase.

DNA Pol II

• It is involved in DNA repair, not replication.

Exonuclease Activity

• Exonuclease activity in DNA polymerases allows for the removal of incorrectly incorporated nucleotides.

Linking Okazaki Fragments

• DNA ligase joins the Okazaki fragments together on the lagging strand.