Expression of mRNA

The expression of mRNA from DNA involves transcription of one strand of DNA (the mRNA coding strand that is the complementary sense of mRNA). Following initiation of transcription, RNA is polymerized with a DNA-dependent RNA polymerase using Watson-Crick base-pairing rules (except that in RNA, U is found in place of T). Although similar in broad outline, many details of the process differ between prokaryotes and eukaryotes. One major difference is that the bacterial enzyme can associate directly with bacterial DNA and the enzyme itself can form a pre-initiation complex and initiate transcription. In eukaryotes, a large number of auxiliary proteins assembling near the transcription start site are required for initiation of transcription, and RNA polymerase can only associate with the template after these proteins

HSV DNA

Origin of replication

V ATP

V

Single stranded DNA binding protein

OCXXXX

Polymerase complex

Helicase/primase

Polymerase complex

Helicase/primase

Polymerase complex

  1. 13.2 Initiation of HSV DNA replication. This process is virtually identical to that occurring in the cell except that virus-encoded enzymes and proteins are involved. The initial step is denaturation of the DNA at the replication origin with origin binding protein. Following this, the helicase— primase complex and ssDNA-binding proteins associate to allow DNA polymerase to begin DNA synthesis. Ori=origin of replication; A/T=AT-rich sequence.
  2. The process of transcription termination also differs significantly between the two types of organism.

Prokaryotic transcription

Regions of prokaryotic DNA to be expressed as mRNA are often organized such that a message is transcribed from which two or more proteins can be translated. The ability of bacterial RNA polymerase to transcribe such mRNA is often controlled by the presence or absence of a DNA-binding protein, called a repressor. The DNA sequence to which the repressor can bind is called the operator and the genes expressed as a single regulated transcript are called operons. This is shown schematically in Fig. 13.3. The operon model for bacterial transcription was first proposed in the early 1960s by Jacob, Monod, and Wollman from their genetic analyses of mutants of E. coli unable to grow on disaccharide lactose. Since then this operon model has been shown to be valid for a large number of prokaryotic transcriptional units.

In addition to organization into operons, prokaryotic gene expression differs from that of eukaryotes as a result of a fundamental structural difference between the cells: the lack of a defined nucleus in prokaryotes. In prokaryotic cells, transcription takes place in the same location and at the same time as translation. This coupling of the two events suggests that the most efficient regulation of gene expression in these cells will be at the level of initiation of transcription. The operon model also takes this into account.

Prokaryotic RNA polymerase

The DNA-dependent RNA polymerase of prokaryotic cells, especially that of E. coli, is well studied. The enzyme shown in Fig. 13.4 contains five subunit polypeptides: two copies of a, one of P, one of P', one of O, and one of ro. The functions of all the subunits except ro are known quite precisely. The core enzyme, which can carry out nonspecific transcription in vitro, consists of the P' subunit for DNA binding and the two O subunits and the P subunit for initiation of transcription and for interaction with regulatory proteins. The addition of the O subunit creates the holoenzyme that transcribes DNA with great specificity, since this subunit is responsible for correct promoter recognition. It is the holoenzyme that is active in vivo for initiation of transcription.

Regulatory gene

Unbound operator Promoter \ Structural genes

Regulatory gene

Unbound operator Promoter \ Structural genes

Pi

Plac lacZ

lacY

Lactose permease

Thiogalactoside transacetylase

Fig. 13.3 The E. coli lac operon. The promoter is always "on," but normally the lac repressor (i) is bound to the operator that blocks transcription. The repressor can be inactivated by addition of lactose. The operator is also sensitive to cAMP levels as explained in the text. All the genes controlled by this operon are expressed as a single mRNA that can be translated into three separate proteins due to internal ribosome initiation.

Thiogalactoside transacetylase

Subunit Size #/molecule Function

a 36.5 kd 2 Chain initiation and interaction with regulatory proteins

ß

151 kd

1

Chain initiation and elongation

ß'

155 kd

1

DNA binding

G

70 kd

1

Promoter recognition

œ

11 kd

1

Helps assemble and stabilize the complex

Fig. 13.4 The bacterial RNA polymerase molecule. The enzyme is made up of six subunits with different functions. The complete enzyme is called the holoenzyme.

The prokaryotic promoter and initiation of transcription

The DNA to which the RNA polymerase holoenzyme binds to begin transcription looks very much like its eukaryotic counterpart. Consensus sequences are present at specific locations upstream from the start site of transcription. A sequence with the consensus TATAAT is found at -10 and a sequence TTGACA at the -35 position. The former sequence is often called the Pribnow box after its discoverer and is similar in function to the TATA box of eukaryotes. The RNA polymerase holoenzyme binds to the promoter, causing a transcription bubble to form in the DNA. Just as in eukaryotes, transcription begins with a purine triphosphate and chain elongation proceeds in the 5' to 3' direction, reading the DNA template from the antisense strand in the 3' to 5' direction. The polymerase catalyzes incorporation of about 10 nucleotides into the growing mRNA before the O subunit dissociates from the complex. Thus, O is required only for correct initiation and transcription of the RNA chain's first portion.

Control of prokaryotic initiation of transcription

As mentioned earlier, the bacterial RNA polymerase holoenzyme will form a transcription complex and begin to copy DNA, given the presence of a correct promoter sequence. Since the strategy of prokaryotic regulation dictates that gene expression be regulated at the level of this initiation, many inducible genes (genes whose expression goes up or down with given cellular conditions) have the general structure of the operon diagrammed in Fig. 13.3. Binding of the repressor protein to the operator sequence of DNA, positioned at or immediately downstream of the initiation site, effectively provides a physical block to progress of the RNA polymerase. The repressor—operator combination acts, in effect, like an "on—off' switch for gene expression, although it should be understood that this binding is not irreversible and that there is some finite chance of transcription taking place even in the "off" state.

Presence of the appropriate inducing molecule, such as the metabolite of lactose responsible for inducing the lac operon, will cause a structural change in the repressor such that it can no longer bind to the operator. In cases such as the tryptophan operon, the repressor protein assumes the correct binding conformation only in the presence of the co-repressor (e.g., tryptophan). The overall situation is that regulated prokaryotic gene expression takes place unless the binding of a protein that blocks movement of RNA polymerase prevents it.

Enhancement of prokaryotic transcription is also seen. Using the example of operons for the genes required to utilize unusual sugars such as lactose, upregulation of gene expression can be observed. In this case, the response involves a system that can "sense" the amount of glucose presented to the cell and thus the overall nutritional state of that cell. Since the enzymes that metabolize glucose (the glycolytic pathway) are expressed constitutively (unregulated) in most cells, the availability of this sugar is a good signal for the cell to use in regulating the expression of enzymes for the metabolism of other sugars. The level of glucose available to the cell is inversely proportional to the amount of 3',5'-cyclic adenosine monophosphate (cAMP) within the cell. This nucleotide can interact with a protein called the cyclic AMP receptor protein (CRP). A complex of cAMP-CRP binds to a region of DNA just upstream of the promoter but only in genes that are sensitive to this effect. When the complex binds, the DNA is changed in such a way that the rate of transcription is raised many fold. If the repressor protein is the "on—off switch of this gene, then the cAMP-CRP complex is the "volume control" fine-tuning transcription as metabolic need arises. This regulation of the rate of transcription by the level of glucose is called catabolite repression.

Termination of prokaryotic transcription

Bacterial RNA polymerase terminates transcription by one of two means: in a p-dependent or p-independent fashion. The difference between these two involves the response of the system to the termination factor (p factor) and structural features near the 3' terminus of the

RNA.

In the case of p-dependent termination, the mRNA being transcribed contains, near the intended 3' end, a sequence to which the p factor binds. The protein p is functional as a hexamer and acts as an ATP-dependent helicase to unwind the product RNA from its template and terminate polymerization.

For p-independent termination, the sequence near the intended 3' terminus of the transcript contains two types of sequence motifs. First, the RNA transcript contains a GC-rich region that can form a base-paired stem loop structure. Immediately downstream from this feature is a U-rich region. The presence of the GC-rich sequence slows progress of the polymerase. The stem loop that forms interacts with the polymerase subunits to further halt their progress. Finally, the AU-rich sequences melt and allow the transcript and template to come apart, terminating transcription.

Eukaryotic transcription

The promoter and initiation of transcription

In eukaryotes, all transcription occurs in the nucleus except for that taking place in organelles. RNA polymerase II (pol II) is "recruited" into the pre-initiation complex formed by association of accessory transcription-associated factors assembling at the site where the transcript is to begin; the process is outlined in Fig. 13.5. Transcription initiates in a "typical" eukaryotic promoter at a sequence of 6—10 bases made up on A and T residues (the TATA box), which occurs about 25 bases upstream (5') of where the mRNA starts (cap site). The proteins making up this pre-initiation complex make a complex just large enough to reach from this region to

1. TF IID binds

"TATA" box

TF IID

TF IID

  • 25 - 30 bp
  • 25 - 30 bp mRNA Cap Site

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