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coli promoters. These include promoters for genes involved inJryptophan, Zactose, and arabinose metabolism. Thesequences vary from one promoter to the next, butcomparisons of many promoters reveal similaritiesin the —10 and —35 regions. The consensus sequences of the —10 and —35 regions are shown atthe bottom. The -10 region is often called the Pribnow box, after David Pribnow, the investigator whofirst recognized it in 1975. All sequences shown arethose of the coding (nontemplate) strand and read5'—»3', left to right, as is the convention in representations of this kind. The spacer regions containvariable numbers of nucleotides (N).
Only the firstnucleotide coding the RNA transcript (at position+1) is shown.SpacerNtrptRNATyr]lac- 1 0 Region] SpacerTTAACTN7N 161 TATGAT |N7N 171 TATGTT |N617| RNA start+1ArecA[WttMfAJN 16I TATAAT |N7AaraB, A, D[OTGACG j ~ N 1 8| TACTGT |N6| AConsensussequenceTATAATMany independent lines of evidence attest to the functional importance of these sequences. Mutations that affect the function of a givenpromoter usually involve one of the base pairs in the - 3 5 or -10 region. Natural variations in the consensus sequence also affect the efficiency of RNA polymerase binding and transcription initiation. Differences of a few base pairs can decrease the rate of initiation by severalorders of magnitude, providing one means by which E.
coli can modulate the expression of different genes. In addition, specific binding ofRNA polymerase to these sequences has been directly demonstrated invitro (Box 25-1).Chapter 25 RNA Metabolism861RNA Polymerase Leaves Its Footprint on a PromoterBOX 25-1Solution of identical DNA fragmentsradioactively labeled • at one end of one strandFootprinting, a technique derived from principles used in DNA sequencing (see Fig. 12-35), isused to identify the specific DNA sequences thatare bound by a particular protein. A DNA fragmentthought to contain sequences recognized by theDNA binding protein is isolated and radiolabeledat one end of one strand (Fig.
1). Chemical or enzymatic cleavage introduces random breaks in theDNA fragment (averaging about one per molecule).Separation of the labeled cleavage products (broken fragments of various lengths) by high-resolution electrophoresis reveals a "ladder" of radioactive bands. In a separate tube the cleavageprocedure is repeated on the original DNA fragment to which the protein is bound. The proteinprevents cleavage of the DNA in the region towhich it is bound. The second set of cleavage prod-Treat with DNaseunder conditions inwhich each strand iscut once (on average).No cuts can be made inthe area where RNApolymerase has bound.Site ofDNase cut „Isolate labeled DNA fragmentsand denature.
Only labeled strandsare detected in next step.###Separate fragments by polyacrylamidegel electrophoresis and visualizeradiolabeled bands on x-ray film.v vUncut DNAfragmentMissing bands indicatewhere RNA polymerasewas bound to DNADNAmigrationxFigure 1 Footprint analysis of the binding site forRNA polymerase on a DNA fragment.
Separateexperiments are carried out in the presence ( + )and absence (-) of RNA polymerase.Continued on next pagePart IV Information Pathways862Coding strand-Regions bound byRNA polymerase+ C--20— -30— -40ucts is subjected to electrophoresis side by sidewith the products of the original reaction. A hole or"footprint" is revealed in the "ladder" of radioactivebands derived from the protein-containing sample.The hole results from the protection of the DNA byprotein binding, and it defines the sequences recognized by the protein.
The precise location of thisbinding site can be determined by directly sequencing (see Fig. 12-35) the original DNA fragment and including the sequencing lanes (notshown here) on the same gel with the footprint.Footprinting results for the binding of RNA polymerase to a DNA fragment containing a promoterare shown in Figure 2. The polymerase covers 50 to60 base pairs; protection by the bound enzyme isconcentrated in the -10 and - 3 5 regions.— -50Figure 2 Footprinting results of RNA polymerasebinding to the lac promoter (see Fig. 25—5). In thisexperiment the 5' end of the coding strand wasradioactively labeled. The C lane is a control inwhich the labeled DNA fragment is cleaved with achemical reagent that produces a more uniformbanding pattern.RNA polymerase binds to the promoter in at least two distinguishable steps (Fig.
25-6). The holoenzyme first binds the DNA and migrates to the - 3 5 region, forming what is called the "closed complex."The DNA is then unwound for about 17 base pairs beginning at the -10region, exposing the template strand at the initiation site. The RNApolymerase binds more tightly to this unwound region, forming an"open complex" (the name reflects the state of the DNA). RNA synthesis then begins.
The binding of RNA polymerase to promoters is facilitated by the supercoiling (underwinding) of the DNA, which may beone of the reasons why cellular DNA is maintained in an underwoundor supercoiled state.The a subunit is required only to ensure the specific recognition ofthe promoter by the RNA polymerase. Once a few phosphodiesterbonds are formed the or subunit dissociates, leaving the core polymerase to complete synthesis of the RNA molecule.Chapter 25 RNA MetabolismFigure 25-6 Steps in the initiation of transcription by E. coli RNA polymerase.
RNA polymerasebinding to a promoter requires two steps: formationof the closed and open complexes. Messenger RNAsynthesis is almost always initiated with a purine(Pu) nucleotide. N is any nucleoside.863RNA polymerase holoenzyme binds to DNA andmigrates to the promoter.RNA polymerase5"3'Promoter-35 -10Polymerase forms aclosed complex at the-35 region.Some E. coli promoters differ greatly from the standard promotersdescribed above, and recognition of these promoters by RNA polymerase is mediated by different a factors.
An example occurs in a set ofgenes called the heat-shock genes, which are induced (their gene products are made at higher levels) when the cell is under the stress thataccompanies an insult such as a sudden temperature jump. RNA polymerase binds to these promoters when its normal a subunit (designated a70 because it has a molecular weight of 70,000) is replaced witha different a subunit that is specific for the heat-shock promoters (seeFig. 27-3). This distinct a subunit has a molecular weight of 32,000and is therefore called a32. The use of different a factors allows the cellto coordinately express sets of genes involved in major changes in cellphysiology.Polymerase migratesto the-10 region. TheDNA is unwound toform the open complex.xpurine nucleotidetriphosphateITemplatestrandSynthesis ofmRNA begins.Initiation of Transcription Is RegulatedUnder certain conditions and at different developmental stages, thecellular requirements for any given gene product may very greatly.
Toprovide proteins to the cell in the proportions needed, the transcriptionof each gene is carefully regulated. The variation in affinity of RNApolymerase for promoters due to differences in promoter sequences, asdiscussed above, is only one level of control. A variety of proteins bindto sequences in and around the promoter and either activate transcription by facilitating RNA polymerase binding or repress transcriptionby blocking the activity of polymerase. In E. coli, an example of a protein that activates transcription is the catabolite gene activatorprotein (CAP), which increases the transcription of genes coding forenzymes that metabolize sugars other than glucose when cells aregrown in the absence of glucose.
Repressors, typified by the Lac repressor, are proteins that block the synthesis of RNA at specific genes.In the case of the Lac repressor, RNA synthesis is blocked at the genesfor enzymes involved in lactose metabolism when lactose is unavailable. Because transcription is the first step in a complicated and energy-intensive pathway leading to protein synthesis, much of the regulation of protein levels in both bacterial and eukaryotic cells is directedat transcription initiation. In Chapter 27 we will describe many mechanisms by which this is accomplished.Eukaryotic Cells Have Three Kinds of RNA PolymerasesThe transcriptional machinery in the nucleus of a eukaryotic cell ismuch more complex than that in bacteria.
Eukaryotes have three different RNA polymerases, designated I, II, and III. Each has a specificfunction and binds to a different promoter sequence. RNA polymerase I(Pol I) is responsible for the synthesis of only one type of RNA, aPR-The cr subunit isreleased aspolymeraseproceeds beyondthe promoter.864Part IV Information PathwaysFigure 25-7 The consensus sequences of somecommon elements in promoters used by eukaryoticRNA polymerase II, derived from a comparison of100 promoters of this type.
A transcription factor(TFIID) binds at the A=T-rich sequence called aTATA box, facilitating the binding of the polymerase. This sequence is commonly found about 25base pairs before the RNA start site. Two otherelements are also sometimes present, found somewhere between -110 and -40: the CCAAT box andGC box are binding sites for other transcriptionfactors that affect polymerase function. Other sequences, some quite distant in the DNA, can affecttranscription (Chapter 27).
Eukaryotic promotersare more variable than their bacterial counterparts,and some RNA polymerase II promoters lack all ofthe sequences shown. As in Fig. 25-5, the sequences are those in the coding (nontemplate)strand.-40-110-25GGCCAATCTGGGCGGTATAAAACCAATboxGCboxTATAbox+1RNA startpreribosomal RNA transcript that contains the precursor for the 18S,5.8S, and 28S rRNAs (see Fig.
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