I.

The Central Dogma

How is the information in DNA introduced into protein sequence?

The Phage Group - used bacteriophages (bacterial viruses) as a model system to study
this problem. Phages were chosen because they are the simplest "organisms" capable of
replicating their genetic material.

A. Ribosomal RNA (rRNA) was shown to be important for protein synthesis,

although production of new rRNA was shown not to be required for production of phage
proteins. After infection of 15N- and 13C-labeled E. coli with a virus, the rRNA still
contained 15N and 13C.

B. Messenger RNA (mRNA) was shown to be involved in gene expression:

• Cohen (1948) infected E. coli with phage in the presence of a brief pulse of 32P
(i.e. the 32P was taken away after a brief exposure at the time of infection - this
method will trace what happens to the 32P during the very early events of phage
infection). It was found that the 32P was incorporated during the early stages of
infection into an unstable RNA.
• Jacob and Monod (1961) proposed that the unstable RNA was mRNA - a genetic
copy of the coding sequences in the phage DNA.
• Spiegelman (~1961) showed that phage DNA will hybridize to the unstable RNA,
confirming that the information in the sequence of the unstable RNA is derived
from the phage DNA.

C. Crick proposed an "adapter" molecule.

It must be able to read the sequence in the mRNA and at the same time "know" which
amino acids are encoded in the message - i.e. this adapter can translate the language of
nucleic acids into the language of proteins. The adapter was later shown to be transfer
RNA (tRNA).

The Central Dogma summary:

Genes in DNA are transcribed into mRNA sequences. The information in mRNA is
translated into protein sequence in a process requiring rRNA and the adapter tRNA.

II. Gene Structure

A. Prokaryotes vs. Eukaryotes (Figure 5.2)

1. In prokaryotes, genes tend to be clustered in coordinately-regulated groups called

operons. The genes are transcribed together on a single transcript and each protein within
the cluster is translated separately. Prokaryotes can "couple" transcription and translation
- i.e. a mRNA being transcribed can begin being translated even before transcription is
complete.

2. In eukaryotes, genes are not clustered in operons. In addition, eukaryotic genes often
contain non-coding introns ("intervening sequences") interspersed among the coding
regions (exons). During RNA processing, introns are removed from RNA transcripts and
the exons are spliced together. Mature mRNA, after being transcribed and processed in
the nucleus, is transported into the cytoplasm where translation occurs. Because
transcription and translation occur in different "compartments" in eukaryotes, "coupling"
of these two processes is not possible.

III. Transcription in Prokaryotes (E. coli)

Transcription involves the construction of an RNA copy of the genetic information in a

DNA template

A. Requirements:

• 1. Substrate ribonucleotide triphosphates (NTPs): ATP, CTP, GTP, UTP

• 2. A template DNA to be transcribed
• 3. RNA polymerase to construct the RNA

B. Stages:

1. Initiation

• a. RNA polymerase consists of 5 subunits: two copies of alpha, and one copy each
of beta, beta' and sigma. The "apoenzyme" contains all subunits except sigma, and
it can construct RNA initiating at any point in a DNA template. The complete
"holoenzyme" contains all five subunits and it can initiate RNA synthesis only at
fixed locations on the DNA. The role of sigma is therefore in the selection of
initiation sites on the DNA - i.e. it tells the RNA polymerase where to start.
• b. Promoter - definition - a site on the DNA to which RNA polymerase can bind
and begin transcription. The sigma subunit recognizes these sequences:

The E. coli "consensus" promoter sequence:

-35 -10 +1
TTGACA (~17 nucleotides) TATAAT (~7 nucleotides) A/G
"Pribnow Box" start
• Promoters vary in their "strength." Strong promoters are recognized by RNA
polymerase frequently - they have sequences close to the consensus sequence.
Weak promoters are not recognized as frequently due to disparity from the
consensus.
Directionality - the product RNA is produced in the 5'--->3' direction. Either
strand of the DNA double helix can contain genes that are transcribed.
2. Elongation -

only requires the RNA polymerase apoenzyme. After about 8 nucleotides are
incorporated into the nascent RNA, the sigma subunit detaches from the RNA
polymerase - it has already fulfilled its task of recognizing the promoter.

3. Termination -

• a. rho-independent - signals in the nascent RNA indicate that transcription should

stop. The signal is an inverted repeat in the RNA sequence followed by a stretch
of several consecutive Us. An inverted repeat in single-stranded RNA is capable
of folding into a stem-loop structure by base-pairing interactions.
• b. rho-dependent - RNA polymerase pauses at specific sequences called "rho
sites"; a protein called rho then binds the RNA polymerase and this causes the
polymerase to fall off the DNA template.

IV. Transcription in Eukaryotes

Eukaryotic transcription is generally more complex than prokaryotic transcription.

A. Requirements:

• 1. Substrate ribonucleotide triphosphates (NTPs): ATP, CTP, GTP, UTP

• 2. A template DNA to be transcribed, containing a promoter. The definition of a
promoter in eukaryotes is looser - it is a sequence of DNA that must be fixed
relative to the transcription start site.
• 3. Many additional protein factors. Some of these bind the DNA before the RNA
polymerase and function in recruiting the RNA polymerase
• 4. A specific RNA polymerase. Eukaryotes have three different types:
o a. RNA polymerase I - transcribes the large rRNA genes
o b. RNA polymerase II - transcribes mRNA genes
o c. RNA polymerase III - transcribes small RNAs - e.g. tRNAs, small
rRNAs
• RNA polymerases I and III are found in the nucleolus, a region of the nucleus rich
in rRNA. RNA polymerase II is found in the nucleoplasm.
• 5. Macromolecules involved in processing, e.g. enzymes

B. mRNA synthesis and processing

• 1. Initiation and elongation: two sequences are recognized during initiation - the
TATA box located at -25 which is bound by TATA binding protein and the CAAT
box located at -60 to -80 which is bound by CAT binding protein. RNA
polymerase subsequently binds and transcription begins. As in prokaryotes, the
first base is usually an A or G. Processing of the mRNA begins while the
transcript is being made.
• 2. Capping - a "cap" is added to the 5' end of eukaryotic RNA transcripts. Capping
enzymes attach a methyl group to the 2' hydroxyl group of the 5' terminal base of
the transcript and attach a 7-methyl-guanosine "cap" to the 5' triphosphate end via
a 5'--->5' linkage.
• 3. Removal of 3' sequences - the 3' ends of nascent transcripts are clipped off.
• 4. Addition of a poly-A tail - an ATP-dependent poly-A polymerase enzyme adds
several hundred adenosine residues to the 3' end of the transcript.
o a. How can this be exploited? The presence of the poly-A tail means that
we have valuable sequence information about the 3' end of eukaryotic
mRNAs. Isolation of mRNA can be accomplished by using oligo- dT
columns - a column with covalently attached oligo-dT can hybridize to
eukaryotic mRNAs by complementary base pairing with the poly-A tails.
Other RNAs, lacking a 3' poly-A tail, will not bind the oligo-dT and can be
easily washed away. Subsequently, the mRNA can be eluted from the
column.
o b. Suppose poly-A is not added - without a poly-A tail, intron splicing will
not occur, and the mRNA will not be transported out of the nucleus.
• 5. Removal of introns - introns are cut out of mRNA and the remaining exons are
spliced together. T. Czech (1980s) discovered that an intron in the rRNA of the
protozoan Tetrahymena could remove itself from a transcript without the
assistance of any proteins. The only requirement was guanosine or GTP. The work
was significant because it demonstrated that RNA could act in a functional role,
overturning a long-held belief that RNA was simply structural material or a
conveyor of genetic information.
• 6. Export to the cytoplasm - processed transcripts travel out of the nucleus and
enter the cytoplasm where they will be translated.