Question

1. Describe in as much detail as possible DNA replication OR protein translation. Use the proper terms for the enzymes (proteins) involved. 2. Define: mutations, spontaneous mutations, induced mutations. 3. Compare the design of negative selection and positive selection plating to the identification of bacterial mutants. 4. How is Ti plasmid used in rDNA 5. Describe the structural characteristics of plasmids and explain the roles they play in microbial cells.

Answer 1

Answer=

1.The process of DNA replication as follows:

The process of synthesis of identical two copies of DNA from a single double stranded DNA molecule is called as DNA replication.

DNA replication completed in three steps:

1.Initiation - it involves the recognition of the position on a DNA molecule where replication will begin.

The proteins or enzymes involved are:

DnaA

Single stranded binding protein(SSB)

DnaB (helicase)

DnaC

DnaG (primase)

The sequence of event occur during initiation is:

1.Binding of DnaA protein to OriC (point where initiation starts)

2.Loading of DNA helicase which opens helix.primase binds to form primosome.

3.Synthesis of RNA primer.

5.Initiation of DNA polymerization by DNA polymerase.

2.Elongation - Involves the events occuring at the replication fork ,where the parent nucleotides are copied.

enzymes required for chain elongation are:

DNA polymerase

SSB

DNA gyrase

DNA ligase.

DNA polymerase catalyze the step by step addition of deoxyribonucleotide units to a DNA chain.The chain elongation reaction catalyzed by DNA polymerases in a nucleophilic attack by the 3'-hydroxyl group of the primer on the innermost phosphorous atom of the deoxyribonucleoside triphosphate.

A phosphodiester bond forms.

DNA chain elongation proceeds in 5' - 3' direction.

At each growing fork ,one strand is called as leading strand ,is synthesized continuously from a single primer on the leading strand template & grows in 5'-3' direction.

lagging strand ,is synthesized discontinuously. The first round of discontinuous strand synthesis on the lagging strand begins.Short pieces of DNA ,called as okazaki fragments are repeatedly synthesized on the lagging strand template.

These okazaki fragments are connected by DNA ligase.

3.Termination - occurs when the parent molecule has been completely replicated.

enzymes required for termination :

terminus binding protein (Tus)

DNA topoisomerase IV.

Replication terminates at the terminus region containing multiple copies of about 23 bp sequences called as Ter sequences.

Ter sequences acts as recognition sites for protein Tus (terminus utilization substance).