Questions

1) Mechanisms of DNA replication

2) Transcription

3) Processing RNA

4) DNA technology. Polymerase chain reaction. Transgenic organisms.

5) Mechanisms of translation

DNA Replication, Transcription and Translation

1) Mechanisms of DNA replication

Watson and Crick's discovery of DNA structure in 1953 revealed a possible mechanism for

DNA replication. They discovered that when DNA replication occurs, it happens in a

semi-conservative fashion. What this basically means is that each new daugther DNA strand is made up of one half of the original, and the other half is a new strand.

Three proposed models of replication are conservative replication, dispersive replication, and semiconservative

replication.

Semi-conservative model

According to the semiconservative model, after one round of replication, every new DNA double helix would be a hybrid that consisted of one strand of old DNA bound to one strand of newly synthesized DNA.

Replication fork

The point at which the two strands of DNA are separated to allow replication

of each strand.

Let's meet the first important enzyme which make the DNA replication happen.

DNA polymarase .There are three of these enzymes, DNA Polymerase I,II,&III. The one we are interested in is DNA Polymerase III. It has the important task of generating a new DNA strand by adding new nucleotides.

DNA Polymerase IIICore Enzyme

DNA polymerase III is the principal replicative enzyme in E. coli.

DNA Polymerase III

DNA-dependent DNA polymerases are enzymes that synthesize new DNA strands using preexisting DNA strands as templates

DNA Polymerase III is 15 times more active biologically than DNA polymerase I

DNA Polymerase III

Has two activity: • 5’ --> 3’ DNA polymerase activity (synthesis) • 3’ --> 5’ exonuclease proofreading activity

(error correction)

The 3’→5’ direction of the proof reading exonuclease helps in the maintenance of high

replicational fidelity.

DNA polymerase III

is able to add new nucleotides only to the 3’end (with –OH) and does not add to the 5’ end of nucleic acid.

uses primer (small RNA) as a starting point; the primer gives 3’ hydroxyl group for the start of DNA-polymerase work.

DNA strand synthesis occurs only in 5’→3’ direction from the RNA primer.

Next enzymes and proteins for synthesis

Helicase unwinds the template strands with breaking the hydrogen bonds

Topoisomerase creates a small cut in the overwounded DNA, to allow the overwounded DNA to unwind and relax.

SSB proteins (Single Stranded Binding proteins) is for stabilization of replication fork

Primase is for primer synthesis DNA-lygase

Mechanism of synthesis

The DNA strand thus synthesized in the 5’→3’ direction is known as the

leading strand.

Only one strand of DNA is synthesized continuously. The synthesis of the

other strand, the lagging strand involves a complicated mechanism.

Mechanism of synthesis

Okazaki fragments

The lagging strand is produced in small fragments as Okazaki fragments. The synthesis of the lagging strand DNA is also in the 5’→3’ direction using a number of RNA primers for individual stretches

RNA primers are then removed by the DNA polymerase I having a 5’→3’ exonuclease. The gaps are filled by this enzyme.

DNA ligase binds Okazaki fragments

2) Transcription

DNA transcription is the synthesis of RNA from DNA

The most important player in prokaroytic transcription is RNA polymerase.

RNA polymerase

This is an enzyme whose job is to actually make the RNA strand from the one of the DNA strands.

In the process of transcription there are three main stages:

Initiation: the construction of the RNA polymerase complex on the gene's promoter with the help of transcription factors

Elongation: the actual transcription of the majority of the gene into a corresponding RNA sequence

Termination: the stop of RNA transcription and the disassembly of the RNA polymerase complex.

DNA template for transcription and mechanism of process

Processing of RNA

is to generate a mRNA (for protein genes) or a functional tRNA or rRNA from the primary transcript.Types of pre-mRNA processing :

1) 3'-Polyadenylation of mRNA: The poly-A tail contains ~ 250 A residues in mammals,  and ~ 100 in yeasts.

2) 5'-Capping of mRNA: the chemical structure of the "cap" is m7G

3)Splising

DNA technology. Polymerase chain reaction. Transgenic organisms

DNA technology has revolutionized modern science. Recent advances in DNA technology including cloning, PCR, recombinanat DNA technology, DNA fingerprinting, gene therapy, DNA microarray technology, and DNA profiling have already begun to shape medicine, forensic sciences, environmental sciences, and national security.

Transgenic Organisms (genetically modified organism)

A genetically modified organism (GMO) or genetically engineered organism (GEO) is an organism whose genetic material has been altered using genetic engineering techniques.These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one molecule to create a new set of genes. This DNA is then transferred into an organism, giving it modified or novel genes. Transgenic organisms, a subset of GMOs, are organisms which have inserted DNA that originated in a different species.

Transgenesis

is the process of introducing an exogenous gene – called a transgene – into a living organism so that the organism will exhibit a new property.

Transgenesis can be facilitated by liposomes, plasmid vectors, viral vectors, and other.

Recombinant DNA technology

Uses of transgenic organisms:

in toxicology: as responsive test animals (detection of toxicants);

in mammalian developmental genetics; to introduce human genes into other organisms

(particularly human) for the study of disease processes;

in molecular biology, the analysis of the regulation of gene expression;

in the pharmaceutical industry, in biotechnology: as producers of specific proteins;

genetically engineered hormones to increase milk yield, meat production; agriculture (food production)

POLYMERASE CHAIN REACTION

The polymerase chain reaction (PCR) was discovered by Kary Mullis.

He won a Nobel Prize in 1993 and became very "FAMOUS" because of this discovery.

PCR is the method of DNA ampliphication (or increasing of quantity of DNA molecules).

This figure represents the PCR reaction.

DNA profiling (DNA testing)

is used in establishing proof of paternity or identifying siblings. While DNA contains material common to all humans, some portions are unique to each individual. DNA testing can help solve crimes by comparing the DNA profiles of suspects to offender samples.

DNA Paternity Test

One of the most popular and readily available of these tests is the DNA paternity test. During this test the genetic profile of the child is compared to the genetic profile of the alleged father, and if a large enough number of similarities are found, then it can be concluded that the alleged father is indeed the biological father of the child.

The accuracy of determining is 99.99%

Translation

is the synthetic of proteins molecules on RNA template

Stages:- Initiation- Elongation- Termination