Question

1.With the mixed copolymer experiment, it was determined that AAC specifies one of the three amino acids: threonine, asparagine, or glutamine. Describe a triplet binding assay experiment that can be used to determine which of the three amino acids is specified by the codon AAC. You will need to include the ingredients that must be added to each tube analyzed in the experiment. Describe the experimental result that would be obtained if AAC specifies asparagine.

2. Briefly describe the difference(s) between Rho-dependent and Rho-independent transcription termination in E. coli. What is Rho? The transcription of some genes is always terminated by Rho-dependent termination, and for other genes by the Rho-independent mechanism. What determines which mechanism of transcript termination will be used for a particular gene?

Answer 1

Ans_1 Nirenberg and and leader developed a triple binding assay . The technique took advantage of the observation that ribosomes , when presented with an RNA sequence of three ribonucleotides will bind to it and form a complex .

The triplet acts as a codon and pairs with anticodon of tRNA . Triplet of known sequence were chemically synthesized. The amino acid to be tested was made radioactive and a changed tRNA was produced

​​​​​​The radioactively charged tRNA the RNA triplet of known sequence and ribosomes are incubated together on a nitrocellulose filter which will retain the ribosomes, but not the other smaller components such as tRNA . IF radioactivity is not retained on the filter , an incorrect amino acid has been tested .

In this way a specific codon assignment was made

Ans_2 Bacteria appear to use two distinct strategies for transcription termination ..one is intrinsic termination and second is rho dependent termination

Intrinsic termination have been thought to promote dissociation of the polymerase by destabilizing the attachment of the growing transcript to the template . IN this case , transcript forms a hairpin structure by forming a complementary base pairing. Intrinsic termination include G.C. rich inverted repeat . Inverted repeat allow the formation of hairpin structure in the transcript which is followed by a U_rich region. The typical distance between the hairpin and the U_rich region in the transcript is 7_9 nucleotides

The second type of termination is RHO_DEPENDENT. This termination requires the activity of a protein called Rho . Rho is an ATP dependent RNA stimulated helicase that disrupts the nascent RNA_DNA complex.It binds to RNA at rut site and translocates along RNA IN 5' TO 3' direction until it reaches the RNA_DNA hybrid in RNA polymerase ,where it release the RNA FORM the DNA. The rut site is rich in C residues and poor in G residues

Answer 2

1.AAC specifies Asaparagine.

Using mixed copolymer experiment mRNA of two different nucleotides.

We need to take any desired extract which aminoacyl tRNA specific to an amino acid, ribosomes, polynucleotide phosphorylase and rest of the translation machinery. To which we need to add our desired nucleotides.

So, now to obtain amino acid specific to AAC codon we need to play with the ratios if we add more of adenine with Cysteine we will get Asparagine in contrast if we add more of Cysteine with Adenine then we will get Histidine so here AAC two molecules of Adenine with one molecule of Cysteine gives Asparagine.

2. Rho dependent termination is a process where the rho protein dissocoates RNA Polymerase and moves it off the template whereas rho independent termination is a process where RNA forms a hairpin structure eventually displaces the enzyme RNA Polymerase and stops the transcription process.

Rho is a protein involved in the transcription termination of prokaryotes. It is a hexamer of identical sub units and each sub unit has a RNA binding domain and ATP hydrolysis domain.

Rho dependent termination occurs when there is specific DNA signals transcribed into RNA called Rho dependent terminators. The fundemental features of Rho dependent terminators are rut site which is a proximal Rho bindign site and and terminal zone as a distal sequence. So, genes which recieve these specific DNA signals will terminate in Rho Dependent manner.

Rho independent termination occurs when there is a series of 'U' residues in the transcribed RNA and ahead of this there is GC rich self complementary with several intervening sequences. Such self complementary sequences when synthesized complement to each other and form a stem loop structure which is thought to interact with RNA Polymerase causing a pause and terminate the transcription.