The synthesis of mRNA (or pre-mRNA) in the nucleus and its subsequent transport to the

cytoplasm can be documented by pulse labeling experiment, pulse-chase labeling experiments,

and autoradiography (fig. 109). If a cell is exposed to a radioactive RNA precursor (such as
[3H]uridine or [3H]cytidine for a few minutes, and the intracellular location of the incorporated
radioactive is determined by autoradiography, almost all the nascent labeled RNA is found in the
nucleus (fig. 10.9a). if on the other hand, the short exposure (pulse) to the labeled RNA
precursor is followed by a period of growth in non radioactivity is found transported to the
cytoplasm (fig. 10. 9b). (Pulse chase experiments are done by either (1) sedimenting the cells
from the radioactive medium or (2) adding a vast excess of nonradioactive precursor so as to
dilute the radioactive precursor to neglibible concentrations.

Strong evidence for an RNA intermediary protein synthesis also resulted from studies on
T2 phage infected E. coli cells. The phage proteins were thrown to be synthesized on ribosomes
present in the cell prior to infection. The specificity determine amino acid sequences of
polypeptides was not therefore an integrate part of ribosome structure E. volkin and L Astrachan
demonstrated that there is a large burst of RNA synthesis shortly after T2 phage infection. And
these short-lived (half-lives of only a few minutes) RNA molecules have nucleotide
compositions like the T2 phage DNA and not like the host DNA. Shortly thereafter, these
unstable RNA molecules were shown to be complementary to segments of strands of DNA in the
phage chromosome. Many different phage mRNA molecules have now been isolated and shown
to direct the synthesis of specific phage proteins by in vitro protein synthesis ( and following
section entided Transiation)

The DNA depent RNA polymerases that catalyze transcription are usually complex,
multimeric proteins. The E. coli RNA polymerase, the most extensively studied of all the RNA
polymerases, has a molecular weight of all about 490.000 and consist of six polypeptides (fig.
10. 10). Two of these are identical; thus the enzyme contains five distinct polypeptides. One of
these subunit, the sigma () factor, is only involved in the initiation of transcription; it does not
have a catalic function. The complete RNA polymerase molecule, called the holoenzyme,
contains two -polipeptides and one polipoptides of each of the following types: , , , . After
initiationof the synthesis of an RNA chain, the -factor is released and chain elongation is
catalized by the so-caled core enzyme, which has the composition 2, , , . The function of
sigma is to recognize and bind RNA polymerase to the correct initiation sites, the promoter sites
(see chapter14), on the DNA. RNA core polymerase (sigma absent) will catalyze RNA synthesis
from DNA templates in vitro, but, on so doing, it initiates at random sites on both strands of
DNA. The holoenzyme (sigma present), on the other hand, initiated in vitro only at sites used in
vivo.

Hundreds of promoters have now been sequenced in E. coli, and these sequnces have
surprisingly little in common. Two short sequences have surprisingly little in common. Two short
sequences within these promoters are sufficiently conserved to be recognized, but even these are
seidom identical in two different promoters. The midpoints of the two conserved to be
recognized, but even these are seidom identical in two different promoters. The midpoint of the
two conserved sequences occurs at about 10 and 35 nucleude pairs, respectivelybefore the
transcription-initiation site. Thus they are often called the 10 sequence and the 35 sequence (also
called the) is TATAAT. The consensus 35 sequence (also called the recognition sequence).

In eukaryotes, there are three different RNA polymerases, I, II, and III. RNA polymerase
I is located in the nucleolus and catalyzes the synthesis of rRNA. RNA polymerases II and III are
present in the nucleoplasm (outside the nucleolus). RNA polymerase III transcribes the genes for
small nuclear RNAs and tRNAs. Its binding sites are located within these genes, rather than on
the 5 or upstream sides of the genes.

RNA polymerase II transcribe the majority of the nuclear structural genes; it is

responsible for pre-mRNA synthesis. Comparisons of the sequences of the promoters or RNA
polymerase II-binding sites of over 200 different eukaryotic genes reveal consensus sequences
located at about 25 and 75 nucleotide-pairs, respectively, before the transcription-start site. The
consensus sequences for the -25 sequence (also called the Ilogness box and the TATA box)
and the -75 sequence (also called the CAAT box) are TATAAAA and GGCCAATCT,
respectively.

The mechanism of RNA synthesis is analogous to DNA synthesis (see chapter 5) except
that (1) the precursor are ribonucleoside triphosphate, (2) only limited segments of single strands
are copied, and (3) the complementary RNA is released from the template as it is synthesized.
Covalent etension occurs, as with DNA synthesis (see chapter 5), by the addition of
mononucleotides to the 3 end of the chain, with the release of pyrophosphate.

The termination of transcription occurs at specific terminator sequences in DNA. Most

prokaryotic mRNAs terminate with the sequences in DNA. Most prokaryotic mRNAs terminate
with the sequence 5UUUUUUA-3, suggesting that the sequence 3-AAAAAAT-5 in the sense
strand of DNA is at least part of the transcription terminator sequence (see chapter 14, Fig
14.11a). some transcription-termination signals require the presence of a protein called rbo,
whereas other do not.

Although only one of the two DNA strands is transcribe in any given region, both strands
of DNA in a chromosome usually participate in transcription, with some mRNAs being
transcribe from one strand and other mRNAs from different genes) being transcribe from the
other strand. Because the two strands of a DNA double helix have opposite polarity, transcription
events using opposite strands as templates will proceed in opposite direction along the DNA
molecule. Event in simple viruses such as (lamda) and r4 (see chapter 12, fig 12.11,
transcription, occurs off both DNA strands (but only rarely in the same region).