Question

principle of genetics: chapter 10:

describe the synthesis of RNA and the experiments which support your description

Answer 1

Solution:

Elliot Volkin and his colleagues reported their analysis of RNA produced immediately after bacteriophage infection of E. coli. They used the isotope ³²P to follow newly synthesized RNA and found that its base composition closely resembled that of the phage DNA, but was different from that of bacterial RNA. This newly synthesized RNA was unstable (short lived); however, its production was shown to precede the synthesis of new phage proteins. Thus, Volkin and his coworkers considered the possibility that synthesis of RNA is a preliminary step in the process of protein synthesis.

RNA Polymerase:

By 1959, Samuel Weiss, had independently discovered  a molecule in rat liver called RNA polymerase, which had the same general substrate requirements as does DNA polymerase, the exception being that the substrate nucleotides contain the ribose instead of deoxyribose form of the sugar. Unlike DNA polymerase, no primer is required to initiate synthesis. The overall reaction summarizing the synthesis of RNA on a DNA template can be expressed as

n(NTP)           (NMP)n + n(PPi)

enzyme RNA polymerase

RNA synthesis

INITIATION:

Once RNA polymerase has recognized and bound to the promoter,double stranded DNA is converted to an open structure, exposing the template strand. The enzyme then proceeds to initiate RNA synthesis, whereby the first 5.-ribonucleoside triphosphate(rNTP), which is complementary to the first nucleotide, is inserted at the start site.No primer is required. Subsequent ribonucleotide complements are inserted and linked together by phosphodiester bonds as RNA polymerization proceeds. This process continues in a 5' to 3' direction.

CHAIN ELONGATION:

After the addition of ribonucleotides to the growing RNA chain, the s subunit dissociates from the holoenzyme, and chain elongation proceeds under the direction of the core enzyme. In E. coli, this process proceeds at the rate of about 50 nucleotides/second at 37°C. The enzyme traverses the entire gene until it encounters a specific nucleotide sequence that acts as a termination signal.

TERMINATION:

Termination sequences, about 40 base pairs in length, are extremely important in prokaryotes because of the close proximity of the end of one gene to the upstream sequences of the adjacent gene.The unique sequence of nucleotides in this termination region causes the newly formed transcript to fold back on itself, forming what is called a hairpin secondary structure, held together by hydrogen bonds. The hairpin is important to termination. In some cases, the termination of synthesis is also dependent on the termination factor, rho (r). Rho is a large hexameric protein that physically interacts with the growing RNA transcript, facilitating termination of transcription. When termination is achieved, the transcribed RNA molecule is released from the DNA template, and the core polymerase enzyme dissociates. The synthesized RNA molecule is precisely complementary to the DNA sequence of the template strand of the gene.