Moreover, deletions that remove part of the trpL region (fig.14.

5) result in increased rates of expression

of the trp operon. The effects of these deletions are independent of repression; the increase occurs in
both the repressed and the derepressed state.

This second level of regulation of the trp operon is called attenuation, and the sequence within
trpL that controls this phenomenon is called the attenuator (fig. 14.10). attenuation occurs by control of
the termination of transcription at a site near the end of the mRNA leader sequence. This “premature”
termination of trp operon transcription occurs only in the presence of tryptophan-changed tRNA-trp and
yields a 140- nucleotide-long leader-sequence transcript (fig.14.10)

The attenuator region has a nucleotide-pair sequence essentially identical to the transcription-
termination signals found at the ends of most bacterial operons (including the trp operon; see fig.11a).
these termination signals contain a GC-rich palin-drome followed by several AT base-pairs. Transcripton
of these termination signals yields a nascent RNA with the potential to form a hydrogen-bonded “bairpin”
structure followed by several U’s (fig.14.11a). when a nascent transcript forms this hairpin structure, it is
belived to cause a conformational the reguration associated RNA polymerase, resulting in bacterium. It
will transcription within the following, or not this loop forigen-bonded {(A;U)n} region of DNA-RNA base
pairing. The nucleotide sequence of the attenuator therefore explains its ability to prematurely terminate
trp operon trascription. But how can this be regulated by presence or absence of tryptophan?

First recall that transcription and translation are coupled in prokaryotes, that is, ribosomes begin
translating mRNAs while they are still being produced by transcription. Thus, events occurring during
translation may also affect transcription.

Second, note that the 162-nucleotide-long leader sequence of the trp operon mRNA (fig.14.10)
contains sequence that can base-pair to form alternate secondary structure. Two of these sequences form
the previously mentioned transcription-termination hairpin (fig.14.11c). this hairpin is formed by base-
pairing between nucleotide sequences 114-121 and 126-134 (nucleotide 1 is at the 5’ terminus). An
alternate secondary structure results form base-pairing between leader sequences 74-85 and 108-119
(fig.14.11b). obviously, only one of these structures can exist at one time, since nucleotides 114-119 are
part of both. Thus, if sequences 74-85 and 108-119 are base –paired, the attenuator transcription hairpin
cannot form.

Third, note that the leader sequence contains an AUG translation-initiation codon, followed by 13
codons for amino acids, followed in turn by a UGA translation-termination codon (fig.14.10). Moreover,
the trp leader sequence has been shown to contain an efficient ribosome-binding site located in the
appropriate position for the initiation of translation at the leader AUG initiation codon. It seems very likely
that a 14-amino-acid-long “leader peptide” is synthesized as diagrammed in fig. 14.10. this putative leader
petide has not yet been detected in vivo, but short peptides of this type are very rapidly degraded in E.
coli¸ so failure to detect it is not unexpected.

Note that the leader peptide contains two contiguous tryptophan residues. The two Trp codons
are positioned such that in the absence of tryptophan (and thus the absence of Trp-TRNA-trp), the
ribosome will become stalled before it encounters the base-paired structure formed by leader sequences
74-85 and 108-119 (fig. 14.11b). this base-pairing precludes the formation of the transcription-
termination hairpin. Thus, in the absence of tryptophan, transcription will continue past the attenuator
into the trpE gene.
 In the presence of tryptophan, the ribosome can translate past the trp codons to the leader-
peptide terminator codon. In the process, it will have to distrubt the base-pairing between leader
sequences 74-85 and 108-119. This, in turn, frees the 114-121 sequences, allowing it to base pair with
126-134 sequences and form the trancription-termination hairpin (fig.14.11c). thus , in the presence of
tryptophan, transcription frequently terminates at the attenuator, reducing the amount of mRNA for the
trp structural genes.

The transcription of the trp operon can be regulated over a range of almost 700-fold by the
combined effects of repression (up to 70-fold) and attenuation (up to 10-fold).

Regulation of transcription by attenuation is not unique to the trp operon. Six operons
(trp,tbr,ilv,leu,phe,and his) are known to be regulated be attenuation. Of these, trp and possibly phe are
also regulated by repression. The bis operon, which has long been thought to be repressible, is now
believed to be regulated entirely by attenuation. Altought minor details vary from operon to operon, the
main features of attenuation are the same for all six operons.

Feedback inhibition and allosteric enzymes

…. In this chapter, we described the mechanism by which the transcription of bacterial genes
coding for enzymes in a biosynthetic pathway is respressed when the end product to the pathway is
present in the medium in which the cells are growing. A second, and more … , regulatory fine-tuning of
metabolism of ten occurs at the level of enzyme activity. The presence of sufficent concerntations of an
end product (such as histidine or tryptophan) of a biosynthetic pathway will frequently result in the
inhibition of the first enzyme in the pathway. This phenomenon is called feedback inhibition or end
product inhibition; it should not be confused with repression (inhibition of enzyme synthesis ). Feedback
inhibition results in an almost instantaneous arrest of the synthesis of an end product when it is added
to the medium.

Feedback inhibition sensitive enzymes have been shown to have an end product binding site (or
site ) in addition to the substrate binding site (or site ). In the case of some multimeric enzymes, the end
product or regulatory binding site is on a different subunit (polypeptide) than the substrate site. Upon
binding the end product, such enzymes are believed to undergo changes in comformation, called allosteric
transitions, that reduce their affinity for their substrates. Proteins that undergo such comformational
changes are usually reffered to as allosteric proteins. Many examples are known, including numerous
feedback inhibition-sensitive enzymes and the repressor molecules discussed in the preceding sections.

Temporal sequences of gene expression during phage infection

Regulation of gene expression during the lytic life cycles of bacteriophages is quite different form
the reveraible on-off switches characteristic of bacterial operons. Instead, viral genes are expressed in
genetically preprogrammed sequences, possibly analogous to the preprogrammed sequences of gene
expression putatively involved in differentiation in higher organisms. Although different bacterial viruses
exhibit variations of the spesific mechanisms involved, a common picture emerges. One set of phage
genes, usually called “early” genes, is expressed immidiately after infection. The product(s) of one or more
of the “early” genes is responsible for turning on the expression of the next set of genes, and so on. Two
to four sets of genes, depending on the virus, are characteristically involved. In all cases studied so far,
the regulation of sequentialgene expressien during phage infection occurs primarily at the level of
transcription.

In there of the most extensively studied bacterial viruses—E.coli phages T4 and T7 and bacillus
subtilis phage SP01- the sequential gene expression is controlled by modifying the promoter specificity of
RNA polymetase, either by the synthesis of a new RNA polymerase (T7) or by phage induced alterations
of the host cell’s RNA polymerase (T4 and SP01)

In phage T7-infected cells, the “early” genes are transcribed by the E. coli RNA polymerase, which
then transcribes all the “late” genes (coding for T7 structural proteins, lysozyme, etc). bacillus subtilis
phage SP01 exhibits a slightly more complex pathway of sequential gene expression , involving three sets
of genes. These three sets of genes are called “early” “middle” and ”late” genes in reference to their time
of expression during the phage reproductive cycle. The SP01 “early” genes are transcribed by the B.
subtilis RNA polymerase. One of the “early” gene-product is a polypeptide that binds to the host cell’s
RNA polymerase, changing its spesificity such that it transcribes the “middle” genes os SP01. Two of the
product of “middle” genes are, in turn, polymerase, further changing its specificity so that it then
transcribes the “late” genes of SP01.

Phage T4 exhibits an even more complex pattern of sequential gene expression, involving several
different modifications of the host cell’s RNA polymerase. Thus, in the case of these bacterial viruses, the
control of the observed sequential gene expression occurs primarily at the level of transcription and
mediated by specific RNA polymerase-promoter sequence inter-actions.