\chapter{Control of Expression of Bacterial Operons}

Different proteins are produced in different amounts within a given cell.
Some proteins, for example those which are involved in synthesizing
nucleotides, 
are needed in large amounts.  Other proteins are needed in
much smaller amounts.  Cellular mechanisms exist to selectively
synthesize those proteins that are needed in large amounts.
Similarly, not every protein that the organism can make is needed all
the time.  For instance, the proteins that digest an energy source
such as lactose are useless
in the absence of lactose.  Organisms have therefore evolved
mechanisms to regulate the amount of protein made in response to
differing environmental conditions.

The amount of a given protein produced by a cell can be expressed as
follows:
\[  \begin{array}{l@{\: = \:}l}
 amt \; of\; protein & (amt \; of \; protein\; made) - (amt \; of\;
protein\; degraded)  \\
 amt \; of\; protein\; made & K  \times  amt  \; of  \; message,  K >
1 \\
amt \; of \; message & (amt \; of \; message \; made) - (amt \;
of \; message \; degraded) 
  \end{array} \]

Many of the mechanisms for  controlling the amount of protein and
message made and degraded are known, and these can be examined
systematically.

Most known bacterial proteins, once made, are relatively stable.
Differences in the amount of protein present are most likely related
to the amount of protein made rather than the amount degraded
\footnote{Watson, p. 382.  Note, however, that only about 10\% of the E.
Coli genome has been defined, and it remains possible that mechanisms
involving differential stability of proteins may play an important
regulatory role for the remaining 90\% of the gene products.}.  Control
of the amount of protein made can be divided into transcriptional
control and translational control.  The amount of protein which is
degraded is governed by {\it post-translational control}.

In bacteria, most control of gene expression is effected at the
transcriptional level.  Consequently, {\sc Genex} concentrates on
transcriptional control.

\section{Control of Transcription}

Synthesis of the mRNA message of a gene or set of genes is regulated
by signals encoded in nucleotide sequences in the DNA.  
Sequences called {\it promoters} specify start sites for the
transcription of mRNA. {\it Terminator} sequences specify
transcription stop sites.  The sequences of specific promoters (and
terminators) can differ among organisms,
and from gene to gene in the same organism, but there are homologies among 
the known sequences\footnote{See Rosenberg and Court.}.
Some sequences interact more effectively with RNA polymerase in
their roles as promoter (or terminator).  Thus regulation of
transcription can occur by varying the efficiency with which polymerase
interacts with these sites.  

Another form of transcriptional control, termed {\it attenuation}, is
exerted during the {\it elongation} phase of transcription.
Attenuation refers to the regulation of whether or not a mRNA molecule
that has been initiated is transcribed to completion.  This is also
dependent on the DNA sequence.

\subsection{Control of Initiation}

The initial interaction of RNA polymerase with a specific region of
DNA is known as {\it initiation}.  If all regions of DNA were equally
attractive to RNA polymerase, transcription would be able to initiate
anywhere along the strand, irrespective of gene boundaries.  This
would result in a large number of ``nonsense'' mRNAs\footnote{i.e. those
which do not code for any protein.} being produced and would be very
wasteful of the cell's energy.  Therefore cells have developed
mechanisms to ensure that initiation only occurs at specific sites
along the DNA strand.

\subsubsection{Characteristics of the promoter}

Initiation occurs at the promoter. It is thought to consist of three
steps: recognition of the promoter site by RNA polymerase; ``firm
binding'' of the polymerase to the DNA; and the actual initiation of
transcription.  By convention, the nucleotide at which transcription
begins is numbered 1.  The promoter region consists of approximately
40 nucleotides preceding the transcription start site (i.e. position
-40).\footnote{If the CAP site is included as part of the promoter, then
the region extends to -80.}

A region located about 35 nucleotides upstream of the transcription
start site is thought to function as the RNA polymerase recognition
site.  It consists of the highly conserved sequence TTG followed by
the slightly less conserved sequence ACA \footnote{``conserved'' refers to
presence across species lines, i.e. conserved in evolution.  It is a
general molecular biological principle that conserved sequences are
functionally important.}.  In some systems it has
been shown that the efficiency of the promoter correlates with how
well the -35 region matches the prototype hexanucleotide TTGACA
\footnote{Rosenberg and Court, p. 325.}.  The sequences surrounding the -35
region do not show definite homology but tend to be AT-rich.  Since AT
pairs are held together by only two phosphodiester bonds, while GC pairs
contain three bonds, it is possible that local melting also plays a
role, allowing the polymerase to unwind the DNA after it has
recognized the TTGACA sequence.

The RNA firm binding site is located in a region approximately ten
nucleotides upstream of the transcription start site.  It is a seven
base pair sequence homologous to the sequence TATAATG.  The T residue
in the sixth position is invariant among known promoters and it is
likely that this nucleotide plays a necessary role in the positioning
of RNA polymerase on the DNA strand.  Again, promoter efficiency
appears to correlate with how well the sequence matches the prototype
\footnote{Rosenberg and Court, p.  330.}.

Transcription of mRNA initiates at a position six to nine base pairs
downstream of the highly conserved T residue in the -10 region.  The
start nucleotide is usually an A or a G.  However, it is impossible to
locate the exact start site without {\it in vivo} or {\it in vitro} data.

A gene whose initiation is normally only controlled by a promoter is
said to be {\it constitutive}.  Its end product is produced in fixed
amounts, regardless of environmental conditions.  There are other
genes whose synthesis can be adapted to reflect growth conditions by
virtue of the interaction of specific {\it regulatory proteins} with a
sequence adjacent to the genes known as an {\it operator}.  A set of
genes transcribed from a single promoter, and whose joint function is
controlled by an operator and a regulatory protein is known as an {\it
operon} (see figure 3.1).  
\begin{figure}
\centering
\setlength{\unitlength}{0.25in}
\begin{picture}(22,3)
\put(2,1){\framebox(2,1){{\it i}}}
\put(8,1){\makebox(1.8,1){{\it p}}}
\put(9.8,1){\dashbox{.25}(.5,1)}
\put(8,1){\line(1,0){1.8}}
\put(8,2){\line(1,0){1.8}}
\put(8,1){\line(0,1){1}}
\put(10.3,1){\makebox(1.9,1){{\it o}}}
\put(10.3,1){\line(1,0){1.9}}
\put(10.3,2){\line(1,0){1.9}}
\put(12.2,1){\framebox(.8,1)}
\put(13,1){\framebox(2,1){{\it x}}}
\put(15,1){\framebox(2,1){{\it y}}}
\put(17,1){\framebox(2,1){{\it z}}}
\put(0,1){\line(1,0){.2}}
\put(.4,1){\line(1,0){.2}}
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\put(4,1){\line(1,0){.8}}
\put(5,1){\line(1,0){.2}}
\put(5.4,1){\line(1,0){.2}}
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\put(7,2){\line(1,0){.2}}
\put(7.4,2){\line(1,0){.2}}
\put(7.8,2){\line(1,0){.2}}
\put(19,1){\line(1,0){.8}}
\put(20,1){\line(1,0){.2}}
\put(20.4,1){\line(1,0){.2}}
\put(20.8,1){\line(1,0){.2}}
\put(19,2){\line(1,0){.8}}
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\put(20.4,2){\line(1,0){.2}}
\put(20.8,2){\line(1,0){.2}}
\end{picture}
\caption{a typical operon.  {\it i} represents the regulatory gene;
{\it p}, the promoter; {\it o}, the operator; and {\it x, y} and {\it
z}, the structural genes of the operon.  Relative sizes of the parts
are not drawn to scale.} 
\end{figure}

\subsubsection{Role of the Regulatory Protein}

A class of proteins called {\it regulatory proteins} is involved in
the control of operons.  The genes which code for regulatory proteins
are called {\it regulatory genes}.  All known regulatory proteins are
either produced constitutively or regulate their own
production\footnote{A possible explanation for this is that if all
regulatory genes were themselves regulated by other regulatory
proteins, it would require an infinite chain of control.}.

Regulatory proteins in bacteria can exhibit both positive and negative
control mechanisms.  In positive control (PC) systems, the presence of
the regulatory protein (the {\it activator}) is necessary for the
expression of the operon.  In negative control (NC) systems, the
operon is expressed unless it is inhibited by the presence of the
regulatory protein (the {\it repressor}).  Most known bacterial operons are
NC.  Some operons are under both positive and negative control.

Regulatory proteins by themselves are not always functional.  If they
were, the operons that they control would be permanently on or off.
Instead, the activity of a regulatory protein is dependent on its
combining with a specific small molecule.  The binding of the small
molecule to the regulatory protein changes the conformation of the
protein, thus affecting its ability to bind to the operator (see
below).

Regulatory proteins are either {\it inducible} or {\it repressible}.
If the binding of the small molecule to the regulatory protein turns
the operon on, then the system is inducible (the small molecule is
called an {\it inducer}).  If the binding of the small molecule turns
the operon off, the system is repressible and the small molecule is
called a {\it corepressor}.

Certain general rules determine whether a system is inducible or
repressible and what the small molecule is likely to be.  An operon
whose genes code for enzymes to digest a certain substance is likely
to be inducible by that substance, since the digestive enzymes are
needed only when the substance is present.  On the other hand, an
operon whose genes code for enzymes to synthesize a certain product is
likely to be repressed by that product, since if the product is
present, there is no need to synthesize it.

The class of operons known as {\it glucose sensitive operons} operate
under both positive and negative control.  Each of these operons
controls the breakdown of a specific sugar.  In the presence of
glucose, none of the glucose sensitive operon's proteins are
synthesized, because the catabolism (breakdown) of glucose lowers the
amount of cyclic AMP (cAMP) within the cell.  cAMP is necessary for
the transcription of all glucose sensitive operons.  It works by
binding the {\it catabolite activator protein (CAP)}, which then binds
to a specific site on DNA (the CAP site).  This acts to increase the
rate at which RNA polymerase binds to the promoter.  Thus the CAP-cAMP
complex is the positive control element for all glucose sensitive
operons.

\subsubsection{The Role of the Operator}

All regulatory proteins act by binding to DNA at specific sites called
{\it operators}.  The operator region is located at or near the
transcription start site, slightly overlapping the promoter.  The
operator sequence must be at least 10-12 nucleotides in length to
avoid the possibility of the same sequence randomly occurring elsewhere
on the chromosome.

The mechanism by which an active repressor molecule inhibits
expression of its associated operon is well understood for several
operons.  Since the operator region includes the transcription start
site, the presence of a bound repressor at the operator physically
blocks the binding of RNA polymerase to the DNA strand.  The action of
activator molecules is not as well understood.  It is thought that
they act by somehow ``opening up'' the DNA helix for the polymerase.

There are thus four models for operon control: PC-inducible,
PC-repressible, NC-inducible, and NC-repressible.  How the operon
reacts to its external environment is dependent on which one of these
four types it is.

\subsection{Control of Elongation}

Transcription termination sites can occur early within, as well as
at the end of, an operon.  These internal termination sites are known
as {\it attenuators}.  In bacterial systems, attenuators have been found in
operons whose end products are involved in the synthesis of amino
acids.  Attenuators are located in a {\it leader region} between the
transcription start site and the structural genes of the operon.
Attenuators regulate gene expression by controlling transcription of
the operon downstream from the attenuator.  The mechanism by which
this occurs takes advantage of the fact that in prokaryotic systems,
translation of the initial segment of the peptide begins while the
later regions are being transcribed.

Analysis of the sequence of the leader region of several operons
revealed that the region contains a potential transcription start site
and termination site.  The termination site is preceded by a segment
rich in the amino acid that is the end product of the corresponding
biosynthetic pathway.  In a bacterial cell lacking this amino acid,
the ribosome translating the leader sequence becomes stalled at these
amino acid codons (due to a lack of charged tRNA molecules) \footnote{tRNA
molecules are said to be charged if they are bound to their
corresponding amino acid, otherwise they are said to be uncharged.}.
The mRNA sequence within the leader region is capable of forming two
alternative secondary structures.  One of these structures is more
stable (and thus thermodynamically favored) when ribosomes have
stalled in the leader region.  This structure does not contain a
functional transcription termination signal.  The other secondary
structure contains a function transcription terminator sequence.
Since transcription and translation proceed cotemporally in
prokaryotic systems, it is thought that the secondary structure in the
stalled message directs the RNA polymerase to continue transcribing
the operon.  Thus transcription of the structural genes of the operon
can be completed, leading to the synthesis of the deficient amino
acid.

\subsection{Control of Termination}

Transcription termination occurs at the end of genes and operons and
is necessary to prevent transcription in one part of the chromosome
from reading through to other genes.  

Comparison of transcription termination site sequences reveals several
common features  \footnote{All the termination data is from Rosenberg and
Court.}.  All contain inverted repeat sequences (palindromes).  These
sequences have the ability to form stable hairpin-loop structures by
base-pairing between nucleotides of the same strand of DNA or RNA, and
might serve for steric recognition.  The termination site is preceded
by a GC-rich region of variable length (from 3-11 consecutive GC
pairs).  Since GC bonds are more stable than AT bonds, this segment
may assist termination by hampering the movement of RNA polymerase
along the template.  Finally, the termination sequences end in a
series of four to eight T residues.  These may enhance release of
the transcript.

As discussed above, most of the control of gene expression in
prokaryotes exists at the transcriptional level.  Other levels of
control are possible, however.  The amount of gene product made can be
affected by the stability of the message ({\it post-transcriptional
control}), by mutations which change the reading of the message ({\it
translational control}), and by stability of the final product ({\it
post-translational control}).

Mutations which affect the stability of the message can only be
hypothesized, since the exact relationship between conformation and
stability is not known.  Similarly, the steady-state level of a
particular protein can be influenced by post-translational
modification (e.g. proteolytic cleavage) which affects its stability.
However, not enough is currently known about these areas for {\sc Genex} to
make any significant use of them. 

\section{Control of Translation}

The amount of protein made from a given message could be influenced by
the relative affinity of ribosomes for that message compared with
other cellular messages.  Thus, a mutation in the ribosome binding site
could theoretically alter gene expression by increasing or decreasing
the rate at which ribosomes attach to and begin translation of a
specific message.  The most extreme example of this is an mRNA
molecule from which the ribosome binding site has been deleted--such a
message would almost never be translated into protein.

Each codon in an mRNA molecule codes for either an amino acid or chain
termination.  Of the 64 possible three-letter codons, three code for
chain termination, and 61 code for amino acids (of which there are
only 20).  Thus several different codons may correspond to the same
amino acid.  However, a mutation which changes even a single
nucleotide of a message may change the protein made from that message.
Ribosomes can only initiate translation at a specific sequence, AUG
\footnote{Much less frequently, GUG.}.   Mutations which alter the nucleotide
sequence of the translated region of the gene can result in the wrong
protein being made (missense mutations), or, if the mutation replaces
a codon for an amino acid with a termination codon, in no protein
being made (nonsense mutations).
