15. Summary
10.15 Summary |
Transcription is regulated by the interaction between trans-acting factors and cis-acting sites. A trans-acting factor is the product of a regulator gene. It is usually protein but can be RNA. Because it diffuses in the cell, it can act on any appropriate target gene. A cis-acting site in DNA (or RNA) is a sequence that functions by being recognized in situ. It has no coding function and can regulate only those sequences that are physically contiguous with it. Bacterial genes coding for proteins whose functions are related, such as successive enzymes in a pathway, may be organized in a cluster that is transcribed into a polycistronic mRNA from a single promoter. Control of this promoter regulates expression of the entire pathway. The unit of regulation, containing structural genes and cis-acting elements, is called the operon.
Initiation of transcription is regulated by interactions that occur in the vicinity of the promoter. The ability of RNA polymerase to initiate at the promoter is prevented or activated by other proteins. Genes that are active unless they are turned off are said to be under negative control. Genes that are active only when specifically turned on are said to be under positive control. The type of control can be determined by the dominance relationships between wild type and mutants that are constitutive/derepressed (permanently on) or uninducible/super-repressed (permanently off).
A repressor protein prevents RNA polymerase either from binding to the promoter or from activating transcription. The repressor binds to a target sequence, the operator, that usually is located around or upstream of the startpoint. Operator sequences are short and often are palindromic. The repressor is often a homomultimer whose symmetry reflects that of its target.
The ability of the repressor protein to bind to its operator is regulated by a small molecule. An inducer prevents a repressor from binding; a corepressor activates it. Binding of the inducer or corepressor to its site produces a change in the structure of the DNA-binding site of the repressor. This allosteric reaction occurs in both free repressor proteins and directly in repressor proteins already bound to DNA.
The lactose pathway operates by induction, when an inducer β-galactoside prevents the repressor from binding its operator; transcription and translation of the lacZ gene then produce β-galactosidase, the enzyme that metabolizes β-galactosides. The tryptophan pathway operates by repression; the corepressor (tryptophan) activates the repressor protein, so that it binds to the operator and prevents expression of the genes that code for the enzymes that biosynthesize tryptophan. A repressor can control multiple targets that have copies of an operator consensus sequence.
Some promoters cannot be recognized by RNA polymerase (or are recognized only poorly) unless a specific activator protein is present. Activator proteins also may be regulated by small molecules. The CAP activator becomes able to bind to target sequences in the presence of cyclic AMP. All promoters that respond to CAP have at least one copy of the target sequence. Binding of CAP to its target involves bending DNA. Direct contact between one subunit of CAP and RNA polymerase is required to activate transcription.
A protein with a high affinity for a particular target sequence in DNA has a lower affinity for all DNA. The ratio defines the specificity of the protein. Because there are many more nonspecific sites (any DNA sequence) than specific target sites in a genome, a DNA-binding protein such as a repressor or RNA polymerase is "stored" on DNA; probably none or very little is free. The specificity for the target sequence must be great enough to counterbalance the excess of nonspecific sites over specific sites. The balance for bacterial proteins is adjusted so that the amount of protein and its specificity allow specific recognition of the target in "on" conditions, but allow almost complete release of the target in "off" conditions.
Gene expression can be controlled at stages subsequent to transcription. Translation may be controlled by a protein that binds to a region of mRNA overlapping with the ribosome-binding site; this prevents ribosomes from initiating translation. RegA of T4 is a general regulator that functions on several target mRNAs at the level of translation. Most proteins that repress translation possess this capacity in addition to other functional roles; in particular, translation is controlled in some cases of autogenous regulation, when a gene product regulates expression of the operon containing its own gene.
The level of protein synthesis itself provides an important coordinating signal. Deficiency in aminoacyl-tRNA causes an idling reaction on the ribosome, which leads to the synthesis of the unusual nucleotide ppGpp. This is an effector that inhibits initiation of transcription at certain promoters; it also has a general effect in inhibiting elongation on all templates.
Attenuation is a mechanism that relies on regulation of termination to control transcription through bacterial operons. It is commonly used in operons that code for enzymes involved in biosynthesis of an amino acid. The polycistronic mRNA of the operon starts with a sequence that can form alternative secondary structures. One of the structures has a hairpin loop that provides an intrinsic terminator upstream of the structural genes; the alternative structure lacks the hairpin. The choice of which structure forms is controlled by the progress of translation through a short leader sequence that includes codons for the amino acid(s) that are the product of the system. In the presence of aminoacyl-tRNA bearing such amino acid(s), ribosomes translate the leader peptide, allowing a secondary structure to form that supports termination. In the absence of this aminoacyl-tRNA, the ribosome stalls, resulting in a new secondary structure in which the hairpin needed for termination cannot form. The supply of aminoacyl-tRNA therefore (inversely) controls amino acid biosynthesis. Attenuation also is controlled in some operons by regulator proteins that bind directly to the leader RNA to assist or inhibit the formation of a terminator hairpin.
An alternative to a regulator protein may be a small RNA that is complementary to a target mRNA; formation of a duplex RNA region may prevent translation by sequestering the initiation site, directly or indirectly. Regulatory RNAs that function by such means are called antisense RNAs.
