Chapter 11 Transcription and RNA Processing

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(No Transcript)
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DNAse activity
means that activity is present
All RNA gets degraded during enzyme preparation
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34.20 C/12 2.85 x 3.448 9.83 3.21 H/1 3.2 x
3.448 11 15.32 N/14 1.09 x 3.44 3.75 9.05
P/31.290 x 3.448 1
5-C, 5-N, 2-H
5-C, 5-N, 3-H, 1-0
5-C, 2-N, 4-H, 2-0
4-C, 3-N, 2-H, 1-0
RibosePhosphate 5-C,8-H,1-P, 5-O
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34.20 C/12 2.85 x 3.448 9.83 3.21 H/1 3.2 x
3.448 11 15.32 N/14 1.09 x 3.44 3.75 9.05
P/31.290 x 3.448 1
5-C, 5-N, 2-H
5-C, 5-N, 3-H, 1-0
5-C, 2-N, 4-H, 2-0
4-C, 3-N, 2-H, 1-0
A/T Avg 5-C 3.5-N 3-H 1-O
5-C, 5-N, 3-H, 1-0
5-C, 5-N, 2-H
5-C, 2-N, 4-H, 2-0
4-C, 3-N, 2-H, 1-0
G/CAvg 4.5-C 4-N 2.5-H 1-O
RibosePhosphate 5-C,8-H,1-P, 5-O
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34.20 C/12 2.85 x 3.448 9.83 3.21 H/1 3.2 x
3.448 11 15.32 N/14 1.09 x 3.448 3.75 9.05
P/31.290 x 3.448 1
A/T Avg 5-C 3.5-N 3-H 1-O
G/CAvg 4.5-C 4-N 2.5-H 1-O
5-C, 5-N, 2-H
5-C, 5-N, 3-H, 1-0
5-C, 2-N, 4-H, 2-0
4-C, 3-N, 2-H, 1-0
RibosePhosphate 5-C 8-H 1-P 5-O
50 GC/50 A/T 4.75 C 3.75 N 2.75 H 1-O
Total Avg 9.75 C 10.75 H 3.75 N 6 O 1-P
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Foundations of a replicative organism
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DNA Discovery by Friedrich Miescher (Swiss,
1844-1895)
He discovered a substance containing both
phosphorus and nitrogen, made up of molecules
that were apparently very large, in the nuclei
of white blood cells Named the substance
nuclein because it seemed to come from cell
nuclei. In 1874 when Miescher separated it into
a protein and an acid molecule. It is now known
as deoxyribonucleic acid (DNA)
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Phoebus Levene
He worked with Albrecht Kossel and Emil Fischer,
the nucleic acid and protein experts at the
turn of the 20th. century
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He conducted experiments that in 1931 suggested
that the four components of DNA occur in
approximately equal ratios He suggested the
possibly that DNA was made of a repeating
tetramer If so, the implication was that the
structure of DNA was too simple and too regular
to contribute to genetic variation attention
thereafter focused on protein as the probable
hereditary substance
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Not only did Levene identify the components of
DNA, he also showed that the components were
linked together in the order phosphate-sugar-base
to form units. He called each of these units a
nucleotide, and stated that the DNA molecule
consisted of a string of nucleotide units linked
together through the phosphate groups, which are
the 'backbone' of the molecule.
Scientist thought that Proteins (made from 20 aa)
were Needed to encode life, not the 4(?) forms of
Nucleotides
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The Tetranucleotide Hypothesis
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The extracts of the separated compounds are
then studied in the ultraviolet
spectrophotometer. The measurement of complete
absorption spectra permits the determination of
the purity of the solutions and at the sameThe
extracts of the separated compounds are
then studied in the ultraviolet
spectrophotometer. The measurement of complete
absorption spectra permits the determination of
the purity of the solutions and at the same time
the quantitative estimation of their contents.
The details of theThe extracts of the separated
compounds are then studied in the ultraviolet
spectrophotometer. The measurement of complete
absorption spectra permits the determination of
the purity of the solutions and at the same time
the quantitative estimation of their contents.
The details of the procedures employed have been
published a. In this manner, adenine,
guanine, uracil, cytosine, and thymine (and also
hypoxantlfine, xanthine, and 5-methylcytosine 4)
can be determined quantitatively in amounts of
240 y. The precision of the method is ?4 for the
purines and even better for the pyrimidines, if
the averages of a large series of determinations
are considered. procedures employed have been
published a. In this manner, adenine,
guanine, uracil, cytosine, and thymine (and also
hypoxantlfine, xanthine, and 5-methylcytosine 4)
can be determined quantitatively in amounts of
240 y. The precision of the method is ?4 for the
purines and even better for the pyrimidines, if
the averages of a large series of determinations
are considered. time the quantitative estimation
of their contents. The details of the procedures
employed have been published a. In this manner,
adenine, guanine, uracil, cytosine, and thymine
(and also hypoxantlfine, xanthine, and
5-methylcytosine 4) can be determined quantitative
ly in amounts of 240 y. The precision of the
method is ?4 for the purines and even better for
the pyrimidines, if the averages of a large
series of determinations are considered.
Erwin Schrödinger published in 1945 a book titled
What is Life? that planted the idea for
searching the secret of life
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Chargaff noted the publication of Avery, MacCleod
and McCarty paper and realized that DNA was the
key to life and set out to prove Levene wrong
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In 1944 Consden et al. showed that it was
possible to separate individual amino acids and
to determine the amino acid composition of
protein hydrolysates by partition chromatography
on paper strips. The method was, in principle,
readily adapted for the separation and
identification of a large number of other
substances, including the purines and pyrimidines
of the nucleic acids (Figure 1), a task carried
out in Chargaffs laboratory by the Swiss
post-doctoral fellow Ernst Vischer 12,
and independently at the Rockefeller Institute by
Rollin Hotchkiss
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Note C not always equal to G
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Apart from not demonstrating equal amounts of the
four bases and thus casting doubt on the validity
of the tetranucleotide hypothesis, certain other
unexpected patterns also emerged the amounts of
purines seemed always to equal those of
pyrimidines (that is, A G C T, or (A
G)/(C T) 1). This had been found by Alfred
Mirsky in 1943, but seems to have been overlooked
by the Chargaff laboratory. More curiously, the
ratios of AG and TC were always similar to each
other whether they were greater or less than 1
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The significance of these relationships was
puzzling and a constant source of comment. At the
end of 1949 Chargaff noted that A comparison of
the molar proportions of the bases reveals
certain striking, but perhaps meaningless, regular
ities. Early in 1950, he wrote It is
noteworthy, although possibly no more than
accidental, that in all desoxypentose nucleic
acids examined thus far the molar ratios of total
purines to total pyrimidines were not far from 1.
More should not be read into these
figures. Later in 1950, apparently as a
last-minute insertion in the paper, Chargaff
wrote It is noteworthy whether this is more
than accidental, cannot yet be said that in
all desoxypentose nucleic acids examined thus far
the molar ratios of total purines and total
pyrimidines, and also of adenine to thymine and
of guanine to cytosine ratios curiously not
actually presented, were not far from 1 2.
The following year, he wrote As the number
of examples of such regularity increases, the
question will become pertinent whether it is
merely accidental or whether it is an expression
of certain structural principles that are shared
by many desoxypentose nucleic acids, despite
far-reaching differences in their individual
composition and the absence of a recognizable
periodicity in their nucleotide sequence. He
then added It is believed that the time has not
yet come to attempt an answer, although clearly
the subject was very much on his mind.
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Why didnt Chargaff predict The Double Helix?
-He didnt want to be wrong like Levene!
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Two reasons why he would have been wrong
1)
2) What if it was some other reason that A/T G/C
ratio was the same????
3) H and X nucleotides?
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Accounting in Saccharomyces cerevisiae
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One of Chargaffs Rules