Biology Department - Arik Dvir - Research

Dr. Dvir's Research: Molecular Mechanisms of Gene Transcription

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The Basic Chemistry of Synthesis and Radio-labeling of a Messenger RNA (mRNA) in vitro

Ribonucleotides and deoxyribonucleotides are the basic building blocks of RNA and DNA. Each nucleotide is made of three essential components - a nitrogen-containing base, a ribose or deoxyribose, and phosphate groups. A base and ribose without the phosphates is called a nucleoside. The nucleotides used in DNA (triphosphates of deoxyadenosine, deoxyguanosine, deoxycytidine, and deoxythymidine) differ from those used in RNA (triphosphates of adenosine, guanosine, cytosine, and uridine). TORBC1

The three phosphates within a nucleotide are designated alpha, beta, gamma (a,b,g). It is the a-phosphate that remains attached to the nucleoside after the nucleotide has been added to the growing RNA chain. TORBC2

When a nucleotide is added to the growing mRNA chain, the b and g phosphate groups are discarded, and the a-phosphate is covalently linked to the end ribose by the formation of a phosphodiester bond. This reaction is catalyzed by RNA Polymerase II. The relatively low amount of mRNA that is synthesized in a single transcription reaction makes visualization of the RNA product very difficult. TORBC3

An efficient way to overcome the visualization problem is to incorporate radioactive phosphate into the mRNA. This can be accomplished by using a nucleotide substrate such as CTP in which the a- phosphate group contains a radioactive isotope of phosphate, ³²P. TORBC4

Messenger RNA synthesis is a promoter and DNA-dependent reaction. Once the polymerase and the general transcription factors have bound at the promoter site and formed the pre-initiation complex, promoter melting follows (opening of the double-stranded structure of the DNA or strand separation). The strand that is used as a template for RNA synthesis is called the template strand. By the principles of base-pairing, the RNA building blocks are added according to the specified sequence. Radioactive alpha phosphates that come from radioactive ribonucleotides result in the “labeling” of the entire mRNA molecule that is formed. The transcripts formed in the reaction are separated by gel-electrophoresis and visualized by autoradiography. TORBC5

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Ribonucleotides and deoxyribonucleotides are the basic building blocks of RNA and DNA. Each nucleotide is made of three essential components - a nitrogen-containing base, a ribose or deoxyribose, and phosphate groups. A base and ribose without the phosphates is called a nucleoside. The nucleotides used in DNA (triphosphates of deoxyadenosine, deoxyguanosine, deoxycytidine, and deoxythymidine) differ from those used in RNA (triphosphates of adenosine, guanosine, cytosine, and uridine).
The three phosphates within a nucleotide are designated alpha, beta, gamma (a,b,g). It is the a-phosphate that remains attached to the nucleoside after the nucleotide has been added to the growing RNA chain.
When a nucleotide is added to the growing mRNA chain, the b and g phosphate groups are discarded, and the a-phosphate is covalently linked to the end ribose by the formation of a phosphodiester bond. This reaction is catalyzed by RNA Polymerase II. The relatively low amount of mRNA that is synthesized in a single transcription reaction makes visualization of the RNA product very difficult.
An efficient way to overcome the visualization problem is to incorporate radioactive phosphate into the mRNA. This can be accomplished by using a nucleotide substrate such as CTP in which the a- phosphate group contains a radioactive isotope of phosphate, ³²P.
Messenger RNA synthesis is a promoter and DNA-dependent reaction. Once the polymerase and the general transcription factors have bound at the promoter site and formed the pre-initiation complex, promoter melting follows (opening of the double-stranded structure of the DNA or strand separation). The strand that is used as a template for RNA synthesis is called the template strand. By the principles of base-pairing, the RNA building blocks are added according to the specified sequence. Radioactive alpha phosphates that come from radioactive ribonucleotides result in the “labeling” of the entire mRNA molecule that is formed. The transcripts formed in the reaction are separated by gel-electrophoresis and visualized by autoradiography.