For the experiment shown Fig. 6.30 and 6.31, what is the advantage to use a labeled incoming nucleotide (α-32P-UTP or α-32P-CTP), not labeled reagent 1 itself? Please draw a diagram to help explain your answer by contrasting the RNA polymerase labeled with α-32P-Reagent (Panel A) vs α-32P-UTP (Panel B).
Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
For the experiment shown Fig. 6.30 and 6.31, what is the advantage to use a labeled incoming
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(b)
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Reagent I
-OCH₂
OH OH
Polymerase-NH₂
Polymerase
PolymeraseNP-o-
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-0
-O-OCH₂
A
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Figure 6.30 Affinity labeling RNA polymerase at its active site.
(a) Structure of one of the affinity reagents (1), an ATP analog. (b) The
affinity-labeling reactions. First, add reagent I to RNA polymerase.
The reagent binds covalently to amino groups at the active site (and
perhaps elsewhere). Next, add radioactive UTP, which forms a
phosphodiester bond (blue) with the enzyme-bound reagent I. This
reaction should occur only at the active site, so only that site
becomes radioactively labeled.
U
14
α
1 2 3 4 5 6
7 8 9 10
Figure 6.31 The B-subunit is at or near the active site where
phosphodiester bonds are formed. Grachev and colleagues labeled
the active site of E. coli RNA polymerase as described in Figure 6.30,
then separated the polymerase subunits by electrophoresis to identify
the subunits that compose the active site. Each lane represents
labeling with a different nucleotide-affinity reagent plus radioactive UTP,
except lanes 5 and 6, which resulted from using the same affinity
11 12 13 14 15 16 17
6.4 Elongation
B²
В
α
147
reagent, but either radioactive UTP (lane 5) or CTP (lane 6). The
autoradiograph of the separated subunits demonstrates labeling of the
B-subunit with most of the reagents. In a few cases, σ was also faintly
labeled. Thus, the B-subunit appears to be at or near the
phosphodiester bond-forming active site. (Source: Grachev et al., Studies on
the functional topography of Escherichia col RNA polymerase. European Journal of
Biochemistry 163 (16 Dec 1987) p. 117, f. 2.)"
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RNA polymerase is a key enzyme in transcription, the process of copying DNA into RNA. To better understand how RNA polymerase works, scientists have developed experiments to label different parts of the enzyme and track its activity. One way to do this is to use a labeled incoming nucleotide, such as α-32P-UTP or α-32P-CTP. This allows scientists to specifically localize the active site of RNA polymerase, where phosphodiester bonds are formed.
In the following section, we will discuss the advantages of using a labeled incoming nucleotide over labeled reagent 1.
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