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Eukaryotic transcription

The Cell: A Molecular Approach. 2nd edition.

❶Addition of an extract of uninfected cells to the transcription reaction with pure RNA polymerase leads to formation of two discrete products identical to the RNA transcripts found inside infected cells immediately after infection. The termination of transcription is signaled by a GC-rich inverted repeat followed by four A residues.

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RNA Polymerase and Transcription
Premature Termination by Attenuation Helps Regulate Expression of Some Bacterial Operons
Prokaryotic DNA Transcription Elongation and Termination

In addition, some E. A sample containing fragments of DNA radiolabeled at one end is divided into two, and one half of the sample is incubated with a protein that binds to a specific DNA sequence within the fragment.

Both samples are then digested with DNase, more The initial binding between the polymerase and a promoter is referred to as a closed-promoter complex because the DNA is not unwound. The polymerase then unwinds approximately 15 bases of DNA around the initiation site to form an open-promoter complex in which single-stranded DNA is available as a template for transcription.

Transcription is initiated by the joining of two free NTPs. As it travels, the polymerase unwinds the template DNA ahead of it and rewinds the DNA behind it, maintaining an unwound region of about 17 base pairs in the region of transcription. The polymerase then more RNA synthesis continues until the polymerase encounters a termination signal, at which point transcription stops, the RNA is released from the polymerase, and the enzyme dissociates from its DNA template.

The simplest and most common type of termination signal in E. Transcription of the GC-rich inverted repeat results in the formation of a segment of RNA that can form a stable stem-loop structure by complementary base pairing. The formation of such a self-complementary structure in the RNA disrupts its association with the DNA template and terminates transcription.

Because hydrogen bonding between A and U is weaker than that between G and C, the presence of A residues downstream of the inverted repeat sequences is thought to facilitate the dissociation of the RNA from its template. Other types of transcription termination signals, in both prokaryotic and eukaryotic cells , depend on the binding of proteins that terminate transcription to specific DNA sequences, rather than on the formation of a stem-loop structure in the RNA.

The termination of transcription is signaled by a GC-rich inverted repeat followed by four A residues. The pioneering studies of gene regulation in E. These investigators and their colleagues analyzed the expression of enzymes involved in the metabolism of lactose, which can be used as a source of carbon and energy via cleavage to glucose and galactose Figure 6.

Otherwise, the cell is able to economize by not investing energy in the synthesis of unnecessary RNAs and proteins. Thus, lactose induces the synthesis of enzymes involved in its own metabolism. On the basis of purely genetic experiments, Jacob and Monod deduced the mechanism by which the expression of these genes was regulated, thereby formulating a model that remains fundamental to our understanding of transcriptional regulation. The starting point in this analysis was the isolation of mutants that were defective in regulation of the genes involved in lactose utilization.

These mutants were of two types: Mutations affecting o resulted in constitutive expression; mutants of i were either constitutive or noninducible.

The function of these regulatory genes was probed by experiments in which two strains of bacteria were mated, resulting in diploid cells containing genes derived from both parents Figure 6. Analysis of gene expression in such diploid bacteria provided critical insights by defining which alleles of these regulatory genes are dominant and which recessive.

Additional experiments in which mutations in o and i were combined with different mutations in the structural genes showed that o affects the expression of only the genes to which it is physically linked, whereas i affects the expression of genes on both chromosome copies in diploid bacteria.

These results led to the conclusion that o represents a region of DNA that controls the transcription of adjacent genes, whereas the i gene encodes a regulatory factor e. The mating of two bacterial strains results in diploid cells that contain genes from both parents. The model of gene regulation developed on the basis of these experiments is illustrated in Figure 6.

Transcription of the operon is controlled by o the operator , which is adjacent to the transcription initiation site. The i gene encodes a protein that regulates transcription by binding to the operator. Since i - mutants which result in constitutive gene expression are recessive , it was concluded that these mutants failed to make a functional gene product.

This result implies that the normal i gene product is a repressor , which blocks transcription when bound to o. The addition of lactose leads to induction of the operon because lactose binds to the repressor , thereby preventing it from binding to the operator DNA. Negative control of the lac operon. The model neatly fits the results of the genetic experiments from which it was derived. In i - cells, the repressor is not made, so the lac operon is constitutively expressed.

Finally, in o c mutants a functional operator has been lost and repressor cannot be bound. Consequently, o c mutants are dominant but affect the expression only of linked structural genes. Confirmation of this basic model has since come from a variety of experiments, including Walter Gilbert's isolation, in the s, of the lac repressor and analysis of its binding to operator DNA. Molecular analysis has defined the operator as approximately 30 base pairs of DNA, starting a few bases before the transcription initiation site.

Footprinting analysis has identified this region as the site to which the repressor binds, blocking transcription. As predicted, lactose binds to the repressor, which then no longer binds to operator DNA. Also as predicted, o c mutations alter sequences within the operator, thereby preventing repressor binding and resulting in constitutive gene expression.

The central principle of gene regulation exemplified by the lactose operon is that control of transcription is mediated by the interaction of regulatory proteins with specific DNA sequences.

This general mode of regulation is broadly applicable to both prokaryotic and eukaryotic cells. Regulatory sequences like the operator are called cis -acting control elements , because they affect the expression of only linked genes on the same DNA molecule.

On the other hand, proteins like the repressor are called transacting factors because they can affect the expression of genes located on other chromosomes within the cell.

The lac operon is an example of negative control because binding of the repressor blocks transcription. This, however, is not always the case; many trans -acting factors are activators rather than inhibitors of transcription. The best-studied example of positive control in E. Glucose is preferentially utilized, so as long as glucose is available, enzymes involved in catabolism of alternative energy sources are not expressed.

For example, if E. Thus, glucose represses the lac operon even in the presence of the normal inducer lactose. Glucose repression generally called catabolite repression is now known to be mediated by a positive control system, which is coupled to levels of cyclic AMP cAMP Figure 6.

The binding of cAMP stimulates the binding of CAP to its target DNA sequences, which in the lac operon are located approximately 60 bases upstream of the transcription start site.

Positive control of the lac operon by glucose. When tryptophan is scarce, however, the entire trp operon is transcribed, including the leader sequence and all the coding sequences for the trp -encoded enzymes. Mutant cells from which the attenuator region has been deleted produce more trp mRNA under all conditions than do normal cells. Attenuation provides a secondary mechanism for controlling expression of the trp operon.

The leader sequence L , which lies between the operator O and the first structural gene E , contains an attenuator site red band at which transcription is more An attenuator site, in effect, is a DNA sequence where a choice is made by RNA polymerase between continued transcription and termination.

When tryptophan is abundant, little initiation of transcription takes place, and virtually all of the transcripts that are initiated terminate at the attenuator. In contrast, when tryptophan is scarce, initiation occurs at a high rate and many RNA polymerase molecules continue transcribing past the attenuator.

Attenuation requires a particular stem-loop structure in the mRNA leader sequence. Formation of this structure depends on the rate of ribosomal translation of the leader sequence, which is engaged by a ribosome soon after it is synthesized. The rate of translation of the leader sequence depends, in turn, on the supply of aminoacyl-tRNAs charged with the amino acids encoded by the sequence. To understand how attenuation of the trp operon occurs, note that four regions in the trp mRNA leader sequence can base -pair Figure a.

The sequence of region 2 can base-pair with either region 1 or with region 3. Similarly, region 3 can base-pair with either region 2 or region 4. Mechanism of attenuation of trp -operon transcription. Four regions shown in color can form alternative stem-loop structures, only one of which leads to attenuation. Region 1 of the leader contains two successive codons for tryptophan. When tryptophan is present in sufficient quantity, the ribosome translates the leader transcript rapidly, melting the base pairs between regions 1 and 2.

Then when regions 3 and 4 of the RNA are synthesized, they pair forming a stem-loop followed by a series of U residues, so that transcription is terminated by the Rho-independent mechanism described earlier Figure b , left. On the other hand, when the supply of tryptophan is low, the ribosome pauses at each tryptophan codon in region 1 of the leader.

Since the ribosome is paused over region 1, region 1 cannot base-pair with region 2, which then is free to base-pair with region 3 as soon as it is synthesized Figure b , right. The resulting stem-loop does not induce termination because it is not followed closely by a string of U residues. In this situation, region 3 is sequestered in the hybrid region and thus cannot base-pair with region 4 when it is synthesized.

Consequently, the terminating RNA structure does not form and RNA polymerase continues transcribing the remainder of the trp operon. This mechanism maximizes attenuation when Trp-tRNA Trp is at a high enough concentration to support rapid translation of tryptophan codons and minimizes attenuation when tryptophan is scarce and the concentration of Trp-tRNA Trp falls.

The small leader peptide translated from the leader sequence is rapidly degraded. A similar attenuation mechanism occurs in several operons encoding enzymes that synthesize other amino acids including phenylalanine, histidine, isoleucine, leucine, and valine.

In each case, the leader sequence of the mRNA contains multiple codons for the amino acid product of a biosynthetic pathway, and an abundance of the amino acid in the cytosol promotes Rho-independent termination within the leader sequence.

Attenuation of other operons that do not encode amino acid biosynthetic enzymes depends on specific RNA -binding proteins that stabilize one alternative RNA secondary structure over another. Since this protein is activated by glucose-induced phosphorylation, attenuation is reduced and expression of the bgl operon is increased in the presence of glucose. In this way, a sequence-specific RNA-binding protein can control gene expression in E. Addition of an extract of uninfected cells to the transcription reaction with pure RNA polymerase leads to formation of two discrete products identical to the RNA transcripts found inside infected cells immediately after infection.

The Rho factor is a hexameric protein around which a to base segment of the growing RNA transcript wraps. Consequently, pausing of polymerase during elongation is thought to be an important component of Rho-dependent termination, as it is in Rho-independent termination. Recent analysis of the E. These transcripts encode more The isolation of E. These cellular proteins function together to prevent termination during transcription of the ribosomal RNA genes.

Protein-binding studies have shown that N protein functions by binding to the nut sequence in newly transcribed RNA. The nut sequence contains two protein-binding regions: Based on binding studies and mutant analyses, the model of N-mediated antitermination shown in Figure has been proposed. N protein binds to the nut box B in the nascent transcript and then interacts with NusA complexed with RNA polymerase.

This complex can move along the DNA for many kilobases, blocking termination at both Rho-dependent and Rho-independent termination sites by an unknown mechanism, so transcription can proceed. After N protein recognizes and binds to the B box in the nut site, it interacts with the NusA-polymerase complex. This example illustrates yet another way that gene expression can be regulated by controlling termination early in a transcription unit. Although considerably less is known about transcription termination in eukaryotes than in bacteria, some basic features and their similarities to and differences from bacterial termination are understood.

Transcription of pre-rRNA genes by RNA polymerase I is terminated by a mechanism that requires a polymerase-specific termination factor.

This DNA -binding protein binds downstream of the transcription unit , unlike the E. In most mammalian protein -coding genes, RNA polymerase II can terminate at multiple sites located over a distance of 0. Recent biochemical experiments suggest that the protein complex that cleaves and polyadenylates the nascent mRNA transcript at specific sequences associates with the phosphorylated carboxyl-terminal domain CTD of RNA polymerase II following initiation see Figure Analogous control mechanisms, involving a choice between chain elongation or termination, occur in some genes transcribed by eukaryotic RNA polymerase II.

We discuss two examples of such regulation next. Currently, transcription of the human immunodeficiency virus HIV genome by RNA polymerase II provides the best-understood example of regulated transcription termination in eukaryotes. Efficient expression of HIV genes requires a small viral protein encoded at the tat locus. In contrast, cells infected with wild-type HIV synthesize long viral transcripts that hybridize to restriction fragments throughout the single HIV transcription unit.

Molecular Cell Biology. 4th edition.

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A summary of Prokaryotic DNA Transcription Elongation and Termination in 's DNA Transcription. Learn exactly what happened in this chapter, scene, or section of DNA Transcription and what it means. Perfect for acing essays, tests, and quizzes, as well as for writing lesson plans.

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Prokaryotic Transcription and Translation. Outline the process of prokaryotic transcription and translation. The prokaryotes, which include bacteria and archaea, are mostly single-celled organisms that, by definition, lack membrane-bound nuclei and other organelles. Elongation and Termination in Prokaryotes. The transcription elongation.

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Upon termination, the process of transcription is complete. By the time termination occurs, the prokaryotic transcript would already have been used to begin synthesis of numerous copies of the encoded protein because these processes can occur concurrently in the cytoplasm. Several mechanisms of regulating transcription termination have been discovered in bacteria and eukaryotes. RNA polymerase itself plays a role in the two principal mechanisms of transcription termination that occur in E. coli.

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Other types of transcription termination signals, in both prokaryotic and eukaryotic cells, depend on the binding of proteins that terminate transcription to specific DNA sequences, rather than on the formation of a stem-loop structure in the RNA.