Prokaryotic transcription

The first step in prokaryotic transcription is the binding of RNA polymerase to DNA at a particular region called 

Model of RNA synthesis by RNA polymerase from the sense strand of DNA. See text for details. 

Segments of the coding strand of conserved regions from various genes showing the common sequence of six bases. The start point for mRNA synthesis is indicated by the heavy letters. The conserved T is underlined. 

The drug rifampin binds to bacterial RNA polymerases and is a useful experimental inhibitor of initiation of transcription. It binds to the P subunit of RNA polymerase, blocking the transition from the chain initiation phase to the elongation phase it is an inhibitor of chain initiation but not of elongation. Actinomycin D also inhibits initiation but does so by binding to DNA. These drugs have limited clinical use because of their toxicity. 

Base sequence of (a) the DNA of the E. coli trp operon at which transcription termination occurs and of (b) the y terminus of the mRNA molecule. The inverted-repeat sequence is indicated by reversed arrows. The mRNA molecule is folded to form a stem-and-loop structure. 

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Enzyme-stabilized single-stranded DNA (known as the open complex) is the first intermediate formed in transcription initiation of RNA polymerases its formation is the rate-limiting step. Designing molecules which bind specifically to the open complex is a strategy for generating potent transcription inhibitors. The redox-stable complex of Cu(I) with 1,2-dimethyl- 1,10-phenanthroline is an example of such a strategy (405). The Cu(I) complex binds specifically to the single-stranded DNA of transcriptional open complexes and is an effective inhibitor of eukaryotic and prokaryotic transcription. 

Important Differences Exist between Eukaryotic and Prokaryotic Transcription... 

Goodrich, J. A., and W. R. McClure, Competing promoters in prokaryotic transcription. Trends Biochem. Sci. 16 394-396, 1991. Two or more bacterial promoters are often found in close proximity and may compete for the binding of RNA polymerase. 

Kustu, S., A. K. North, and D. S. Weiss, Prokaryotic transcriptional enhancers and enhancer-binding proteins. Trends Biochem. Sci. 16 397-401, 1991. First discovered in eukaryotes, enhancers have now been found to exist for a number of prokaryotic genes. 

RNA polymerase II transcribes messenger RNA and a few other small cellular RNAs. Class II promoters are usually defined by their sensitivity to a-amanitin. Like prokaryotic promoters, many class II promoters contain two conserved sequences, called the CAAT and TATA boxes. The TATA box is bound by a specialized transcription factor called TBP (for TATA-Binding-Factor). Binding of TBP is required for transcription, but other proteins are required to bind to the upstream (and potentially downstream) sequences that are specific to each gene. Like prokaryotic transcripts, eukaryotic RNAs are initiated with a nucleoside triphosphate. Termination of eukaryotic mRNA transcription is less well understood than is termination of prokaryotic transcription, because the 3 ends of eukaryotic mRNAs are derived by processing. See Figure 12-9. 

Using 51-nucleotide sequence windows, Nair et al. (1994) devised a neural network to predict the prokaryotic transcription terminator that has no well-defined consensus patterns. In addition to the BIN4 representation (51 x 4 input units), an EIIP coding strategy was used to reflect the physical property (Le., electron-ion interaction potential values) of the nucleotide base (51 units). The latter coding strategy reduced the input layer size and training time but provided similar prediction accuracy. 

Nair, T. M Tambe, S. S. Kulkarm, B. D. (1994). Application of artificial neural networks for prokaryotic transcription terminator predictioa FEBS Lett 346,273-7. 

In prokaryotes, transcription and translation are closely coupled. Several ribosomes can simultaneously translate an mRNA, forming a polysome. 

We have seen how interactions between DNA-binding proteins such as CAP and RNA polymerase can activate transcription in prokaryotic cells (Section 31.1.6). Such protein-protein interactions play a dominant role in eukaryotic gene regulation. In contrast with those of prokaryotic transcription, few eukaryotic transcription factors have any effect on transcription on their own. Instead, each factor recruits other proteins to build up large complexes that interact with the transcriptional machinery to activate or repress Panscription. 

Eukaryotes contain nuclei. Therefore, transcription is separated from translation. In prokaryotes, transcription and translation occur simultaneously. 

Zheng, M. and G. Storz. Redox sensing by prokaryotic transcription factors. Biochem. Pharmacol. 59 1-6, 1999. 

Proteins are synthesized in the amino-to-carboxyl direction, and mRNA is translated in the 5 —>3 direction. The start signal on prokaryotic mRNA is usually AUG preceded by a purine-rich sequence that can base-pair with 16 5 rRNA. In prokaryotes, transcription and translation are closely coupled. Several ribosomes can simultaneously translate an mRNA, forming a polysome. 

Most prokaryotic transcripts originate using adenosine-5 -triphosphate and, to a lesser extent, guanosine-5 -triphosphate at the start site of the growing RNA chain. 

In prokaryotes, most transcription factors belong to the Helix-Turn-Helix family (19). Proteins of this class typically form homodimers, whose tridimensional structure is symmetrical. As a consequence, many prokaryote transcription factors bind to spaced motifs (also called dyads), where each halfmotif is bound by one element of the homodimer. The width of the spacing between the two contact points is transcription factor-specific, and can vary from 0 to 20 nt. Because we are working with bacterial sequences, we will illustrate the pattern discovery step by using a tool dedicated to the detection of spaced motifs dyad-analysis (20) (see Note 9). 

Alifano, P Rivellini, F., Limauro, D., Bruni, C. B., and Carlomagno, M. S. (1991) A consensus motif common to all Rho-dependent prokaryotic transcription terminators. Cell 64, 553-563. 

Transcription in Prokaryotes Transcription in Eukaryotes GENE EXPRESSION... 

In prokaryotes such as E. coli, most of the control of protein synthesis occurs at the level of transcription. (Refer to Section 18.3 for a discussion of the principles of prokaryotic transcriptional control.) This circumstance makes sense for several reasons. First, transcription and translation are directly coupled that is, translation is initiated shortly after transcription begins (Figure 19.8). Second, the lifetime of prokaryotic mRNA is usually relatively short. With half-lives of between 1 and 3 minutes, the types of mRNA produced in a cell can be quickly altered as environmental conditions change. Most mRNA molecules in E. coli are degraded by two exonucleases, referred to as RNase II and polynucleotide phosphorylase. 

S.L. Dove, J.K. Joung, A. Hochschild, Activation of prokaryotic transcription through arbitrary protein-protein contacts, Nature 1997, 386, 627-630. 

At 37 C, an E. coli ribosome can synthesize a 300-residue polypeptide chain in about 20 seconds. This rate is almost exactly the same as that calculated for prokaryotic transcription. mRNA can be translated as fast as it is transcribed because it is possible for many ribosomes to simultaneously translate a single message. Such a complex is called a polyribosome. Under some conditions, as many as 50 ribosomes may be packed onto a single mRNA. 

As in prokaryotic transcription (Fig. 28-4) elongation by RNA polymerase II occurs within a transcription bubble of 20-30 nucleotides in length. Most transcriptionally active DNA is still in the form of nu-cleosomes, which must be unwound as the transcription bubble moves. Details are still uncertain. ... 

As in prokaryotic transcription (Fig. 28-4) elongation by RNA polymerase II occurs within a transcription bubble of 20-30 nucleotides in length.4 7 ]y[ggt transcriptionally active DNA is still in the form of nu-cleosomes, which must be unwound as the transcription bubble moves. Details are still uncertain.7 488 All of the major steps in processing of the pre-mRNA transcripts, which include capping, splicing, 3 -end cleavage, and polyadenylation (Eq. 28-6), are coupled to transcription. This is apparently accomplished, in part, by physical connections of the necessary proteins to the CTD domain of RNA polymerase 11.4 4,3123 While pre-mRNA usually undergoes all of the steps of Eq. 28-6, rRNA and tRNAs are not capped or poly-adenylated and often are not spliced. 

Prokaryotic transcription is catalyzed by RNA polymerase, which is a 470,000-Da enzyme with five types of subunits a, (0, P, P, and o. 

In prokaryotes, transcription is controlled in four principal ways—alternative O factors, enhancers, operons, and transcription attenuation. They will be discussed in turn. 

As seen in prokaryotic transcription, enhancers and silencers are regulatory sequences that augment or diminish transcription, respectively. They can be upstream or downstream from the transcription initiator, and their orientation doesn t matter. They act through the intermediary of a gene-specific transcription-factor protein. As shown in Figure 11.21, the DNA must loop back so that the enhancer element and its associated transcription factor can contact the preinitiation complex. How this looping enhances transcription is stUl unknown. 

Control of eukaryotic transcription includes many of the same concepts seen with prokaryotic transcription. 

How does RNA polymerase know where to begin transcription In prokaryotic transcription, RNA polymerase is directed to the gene to be transcribed by the interactions between the polymerase s O-subunit and sequences of DNA near the start site called promoters. Gonsensus sequences have been established for prokaryotic promoters, and the key elements are sequences at —35 and —10, the latter called the Pribnow box. In eukaryotic transcription, RNA polymerase binds to promoters as well, but there is no O-subunit, although there is a specific subunit, RBP4, that is involved in promoter recognition. 

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