This sequence is called the -10 box (or the Pribnow box). The second, centered near nucleotide -35 often has the sequence TTGACA. This is the -35 box.
On binding to the promoter sequence (Fig. 6.4a), the a factor brings the other subunits (two of a plus one each of j and j') of RNA polymerase into contact with the DNA to be transcribed. This forms the closed promoter complex. For transcription to begin, the two strands of DNA must separate, enabling one strand to act as the template for the synthesis of an RNA molecule. This formation is called the open promoter complex. The separation of the two DNA strands is helped by the AT-rich sequence of the -10 box. There are only two hydrogen bonds between the bases adenine and thymine; thus it is relatively easy to separate the two strands at this point. DNA unwinds and rewinds as RNA polymerase advances along the double helix, synthesizing an RNA chain as it goes. This produces a transcription bubble (Fig. 6.4b). The RNA chain grows in the 5' to 3' direction, and the template strand is read in the 3' to 5' direction (Fig. 6.4).
When the RNA chain is about 10 bases long, the a factor is released from RNA polymerase and plays no further role in transcription. The j subunit of RNA polymerase binds ribonucleotides and joins them together by catalyzing the formation of phosphodiester links as it moves along the DNA template. The p' subunit helps to keep the RNA polymerase attached to DNA. The two a subunits are important as they help RNA polymerase to assemble on the promoter (see discussion of the lac operon below).
RNA polymerase has to know when it has reached the end of a gene. Escherichia coli has specific sequences, called terminators, at the ends of its genes that cause RNA polymerase to stop transcribing DNA. A terminator sequence consists of two regions rich in the bases G and C that are separated by about 10 bp. This sequence is followed by a stretch of A bases. Figure 6.5 shows how the terminator halts transcription. When the GC-rich regions are transcribed, a hairpin loop forms in the RNA with the first and second GC-rich regions aligning and pairing up. Formation of this structure within the RNA molecule causes the transcription bubble to shrink because where the template DNA strand can no longer bind to the RNA molecule it reconnects to its sister DNA strand. The remaining interactions between the adenines in the DNA template and the uracils in the RNA chain have only two hydrogen bonds per base pair and are therefore too weak to maintain the transcription bubble. The RNA molecule is then released, transcription terminates, and the double helix reforms. This type of transcription termination is known as rho-independent termination.
Some E. coli genes contain different terminator sites. These are recognized by a protein, known as rho, which frees the RNA from the DNA. In this case transcription is terminated by a process known as rho-dependent termination.
|| CONTROL OF BACTERIAL GENE EXPRESSION
Many bacterial proteins are always present in the cell in a constant amount. However, the amount of other proteins is regulated by the presence or absence of a particular nutrient. To grow and divide and make the most efficient use of the available nutrients, bacteria have to adjust quickly to changes in their environment. They do this by regulating the production of proteins required for either breakdown or synthesis of a particular compound. Gene expression in bacteria is controlled mainly at the level of transcription. This is because bacterial cells have no nuclear envelope, and RNA synthesis and protein synthesis are not separate but occur simultaneously. This is one reason why bacteria lack the more sophisticated control mechanisms that regulate gene expression in eukaryotes.
Each bacterial promoter usually controls the transcription of a cluster of genes coding for proteins that work together on a particular task. This collection of related genes is called an operon and is transcribed as a single mRNA molecule called a polycistronic mRNA. As shown in Figure 6.6, translation of this mRNA produces the required proteins because there are several start and stop codons for protein synthesis along its length. Each start and stop codon (page 79) specifies a region of RNA that will be translated into one particular protein. The organization of genes into operons ensures that all the proteins necessary to metabolize a particular compound are made at the same time and hence helps bacteria to respond quickly to environmental changes.
The three major factors involved in regulating how much RNA is made are (1) nucleotide sequences within or flanking a gene, (2) proteins that bind to these sequences, and (3) the environment. The human intestine contains many millions of E. coli cells that must respond very quickly to the sudden appearance of a particular nutrient. For instance, most foods do not contain the disaccharide lactose (Fig. 6.7), but milk contains large amounts. Within minutes of our drinking a glass of milk, E. coli in our intestines start to produce the enzyme p-galactosidase that cleaves lactose to glucose and galactose (Fig. 6.7). In general, transcription bubble
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