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There are four major parts in this chapter:
Fig 16-1
b. To Repress Expression
c. To Activate Expression
3.Some Activators Work by Allostery and Regulate Steps after RNA Polymerase Binding:
In some cases, RNA Polymerase binds efficiently unaided and forms a stable closed complex, which does not spontaneously undergo transition to the open complex.
Activator that stimulate this kind of promoter wrk by triggering a conformational change in either RNA Polymerase or DNA. That is, they interact with the stable closed complex and induce a conformational change that causes transition to the open complex.
Fig 16-2
Some proteins interact with each other even when bound to sites well separated on the DNA
Fig 16-3
Fig 16-4
In this example, we also call the DNA-binding protein architectural
proteins.
Antitermination and Beyond: Not All of Gene Regulation Targets Transcription Initiation The bulk of gene regulation takes place at the initiation of transcription in both eukaryotes and bacteria. But regulation is certainly not restricted to that step in either class of organism. In this chapter we will see examples, in bacteria, of gene regulation that involve transcriptional elongation, RNA processing, and translation of the mRNA into protein.
Part Two
Fig 16-5
DNA m-RNA
PlacI
Plac Olac
Protein
Transacetylase -Galactosidase Permease
The activator is called CAP( Catabolite Activator Protein ) .CAP can bind DNA and activate the lac genes only in the absence of glucose. The lac repressor can bind DNA and repress transcrition only in the absence of lactose.
Both CAP and lac repressor are DNA-binding proteins and each binds to a specific site n DNA at or near the lac promoter.
Fig 16-6
CAP and lac repressor have opposing effects on RNA polymerase binding to the lac promoter
1.Lac operator ------the site bound by lac repressor
This 21 bp sequence is twofold summetric and is recognized by two subunits of lac repressor, one binding to each half-site.
Fig 16-7
lac operator overlaps promoter, and so repressor bound to the operator physically prevents RNA polymerase from binding to the promoter.
Fig 16-8
2. CAP CAP binds as a dimer to a site similar in length to the lac operator, but different in sequence and location.
Fig 16-9
At the promoter,where there is no UP-element, a CTD binds to CAP and adjacent DNA instead.
CAP and lac repressor bind DNA using a common structural motif
1.The Same
A. The protein binds as a homodimer to a site that is an inverted repeat or near repeat.
B.Both CAP and lac repressor bind DNA using a helix-turn-helix motif.
One of the two helices in helix-turn-helix domain is the recognition helix that can fits into the major groove of the DNA.
Fig 16-11
The second helix of the helix-turn-helix domain sits across the major groove an makes contact with the DNA backbone , ensuring proper presentation of the recognition helix, and at the same time adding binding energy to the overall protein-DNA interaction.
2. The Difference
Lac repressor binds as a tetramer, with each operator is contacted by a repressor dimer.
Fig 16-13
In some cases, other regions of the protein, outside the helix-turn-helix domain, also interact with the DNA. In many cases, binding of the protein does not alter the structure of the DNA .In some cases, however, various distortions are seen in the protein-DNA complex.
The activity of Lac repressor and CAP are controlled allosterically by their signals
Binding of the corresponding signals alter the structure of these two regulatory proteins
Response to lactose
Absence of lactose
i
p
Lack of inducer: the lac repressor block all but a very low level of transcription of lacZYA .
o
Active
Inactive
Response to glucose
The lac genes provide an example of signal integration: their expression is controlled by two signals, each of which is communicated to the genes via a single regulatorthe lac repressor and CAP, respectively.
regulator (CAP) works together with different repressor at different genes, this is an example of Combinatorial Control. In fact, CAP acts at more than 100 genes in E.coli, working with an array of partners.
Combinatorial
control is a characteristic feature of gene regulation. More complex organismshigher eukaryotes in particular---tend to have more signal integration.
Recall from Chapter 12 that it is the subunit of RNA polymerase that recognizes the promoter suquences.
Promoter recognition
Different factors binding to the same RNA Pol
Many bacteria produce alternative sets of factors to meet the regulation requirements of transcription under normal and extreme growth condition.
Heat
shock--- 32
When E.coliis subject to heat shock, the amount of this new factor increases in the cell, it displaces 70 from a proportion of RNA polymerases ,and directs those enzymes to transcribe genes whose products protect the cell from the effects of heat shock. The level of 32 is increased by two mechanisms: first, its translation is stimulated---that is,its mRNA is translated with greater efficiency after heat shock than it was before; and second, the protein is transiently stabilized.
Bacteriophages
Many bacteriophages synthesize
Fig 16-14
a cascade of factors which allow a defined sequence of expression of different phage genes .
Normal bacterial holoenzyme Express early genes Encode 28 Express middle genes (gene 34 and 33 )
NtrC Has ATPase Activity and Works from DNA Sites Far from the Gene NtrC has separate activating and DNA-binding domains, and binds DNA only when the nitrogen levels are low.
NtrC binds four sites located some 150 base pairs upstream of the promoter
MerR activates transcription by twisting promoter DNA MerR controls a gene called merT, which encodes an enzyme that makes cells resistant to the toxic effects of mercury In the presence of mercury, MerR binds to a sequence between 10 and 35 regions of the merT promoter and activates merT expression.
The merT promoter is unusual. The distance between the -10 and -35 elements is 19bp instead of the 15 to 17 bp typically found in an eddicient 70 promoter. So, these two elements recognized by are neither optimally seperated nor aligned.
Fig 16-15 a
The binding of MerR locks the promoter in the unpropitious conformation in the absence of Hg2+ .
Fig 16-15 b
When Hg2+ is present, MerR binds Hg2+ and undergo conformational change, which twists the promoter to restore it to the structure close to a strong 70 promoter
Just like this :
Fig 16-15 c
Fig 16-15
Some repressors hold RNA polymerase at the promoter rather than excluding it
Repressors work in different ways :
By binding to a site overlapping the promoter, it blocks RNA polymerase binding. (lac repressor) The protein holds the promoter in a conformation incompatible with tanscription initiation.(the MerR case) Blocking the transition from the closed to open complex. Repressors bind to sites beside a promoter, interact with polymerase bound at that promoter and inhibit initiation. (E.coli Gal repressor)
Different from the Lac operon, two activators AraC and CAP work together to activate the araBAD operon expression
Fig 16-18
The magnitude of induction of the araBAD promoter by arabinose is very large , and for this reason the promoter is often used in expression vectors. Expression vectors are DNA constructs in which efficient synthesis of any protein can be ensured by fusing a gene to a strong promoter .
Fig 16-19
The trp operon encodes five structural genes required for tryptophan synthesis. These genes are regulated to efficiently express only when tryptophan is limiting.
There are two layers of regulation involved: (1) transcription repression by the Trp repressor (initiation); (2) attenuation
161 nucleotides of RNA are made from tryptophan promoter before RNA polymerase encounters the first codon of trpE.
Near the end of this leader sequence ,and before trpE , is a transcription terminator, composed of a characteristic hairpin loop in the RNA.
The hairpin loop is followed by 8 uridine residues. At this so-called attenuator , transcription usually stops,yielding a leader RNA 139 nucleotides long.
Fig 16-20
The sequence encoding the leader peptide has a striking feature : two tyrptophan codons in a row.
The fuction of these codons is to stop a ribosome attempting to translate the leader peotide.
Above all , how transcription termination at the trp operon attenuator is controlled by the availability of tryptophan
Fig 16-21
The
1.
Importance
of
Attenuation
2.
3.
4.
Use of both repression and attenuation allows a fine tuning of the level of the intracellular tryptophan. Attenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuation for their regulation. Provides an example of regulation without the use of a regulatory protein, but using RNA structure instead. A typical negative feed-back regulation.
For each operon,one ribosomal protein binds the messenger near the translation initiation sequence of the first genes in the operon, preventing ribosomes from binding and initiating translation. Repressing translation of the first gene also prevents expression of some or all of the rest. The strategy is very sensitive. A few unused molecule of protein L4, for example, will shut down synthesis of that protein and other proteins in this operon.
Fig 16-22
The mechanism of one ribosomal protein also functions as a regulator of its own translation: the protein binds to the similar sites on the ribosomal RNA and to the regulated mRNA
Fig 16-23
Part Four
Bacteriophage is a virus that infects E.coli. Upon infection, the phage can propagate in either of two ways: lytically or lysogenically. A lysogen is extremely stable under normal circumstances ,but the phage dormant within it---the prophage---can efficiently switch to lytic growth if the cell is exposed to agents that damaged DNA . This switch from lysogenic to lytic growth is called lysogenic induction.
Repressor and Cro Bind in Different Patterns to Control Lytic and Lysogenic Growth Repressor bound to OR1and OR2 turns off transcription from PR . And Repressor bound at OR2 contacts RNA polymerase at PRM, activating expression of the cI gene. OR3 lies with PRM; Cro bound there represses transcription of cI.
Another Activator, cII, Controls the Decision between Lytic and Lysogenic Growth upon Infection of a new Host
cII is a transcriptional activator. It binds to a site upstream of a promoter called PRE and stimulates transcription of the cI gene from that promoter.
Transcriptional Antitermination in Development The transcripts controlled by N and Q proteins are initiated perfectly well in the absence of those regulators. But the transcripts terminate a few hundred to a thousand nucleotides downstream of the promoter unless RNA polymerase has been modified by the regulator; N and Q protein are therefore called antiterminators.
Principles of gene regulation. (1) The targeted gene expression events; (2) the mechanisms: by recruitment/exclusion or allostery Regulation of transcription initiation in bacteria: the lac operon, alternative s factors, NtrC, MerR, Gal rep, araBAD operon Examples of gene regulation after transcription initiation: the trp operon, riboswitch, regulation of the synthesis of ribosomal proteins