Catabolite Activator Protein (CAP): An Activator Regulator
When glucose levels decline in E. coli, catabolite activator protein (CAP) is bound by cAMP to promote transcription of the lac operon.
Explain how an activator works to increase transcription of a gene
- Catabolite activator protein (CAP) must bind to cAMP to activate transcription of the lac operon by RNA polymerase.
- CAP is a transcriptional activator with a ligand-binding domain at the N-terminus and a DNA -binding domain at the C-terminus.
- cAMP molecules bind to CAP and function as allosteric effectors by increasing CAP’s affinity to DNA.
- RNA polymerase: a DNA-dependent RNA polymerase, an enzyme, that produces RNA
- operon: a unit of genetic material that functions in a coordinated manner by means of an operator, a promoter, and structural genes that are transcribed together
- promoter: the section of DNA that controls the initiation of RNA transcription
Catabolite Activator Protein (CAP): An Activator Regulator
Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the operator sequences that act as a positive regulator to turn genes on and activate them. For example, when glucose is scarce, E. coli bacteria can turn to other sugar sources for fuel. To do this, new genes to process these alternate genes must be transcribed. This type of process can be seen in the lac operon which is turned on in the presence of lactose and absence of glucose.
When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E. coli. When glucose levels decline in the cell, accumulating cAMP binds to the positive regulator catabolite activator protein (CAP), a protein that binds to the promoters of operons that control the processing of alternative sugars, such as the lac operon. The CAP assists in production in the absence of glucose. CAP is a transcriptional activator that exists as a homodimer in solution, with each subunit comprising a ligand-binding domain at the N-terminus, which is also responsible for the dimerization of the protein and a DNA-binding domain at the C-terminus. Two cAMP molecules bind dimeric CAP with negative cooperativity and function as allosteric effectors by increasing the protein’s affinity for DNA. CAP has a characteristic helix-turn-helix structure that allows it to bind to successive major grooves on DNA. This opens up the DNA molecule, allowing RNA polymerase to bind and transcribe the genes involved in lactose catabolism. When cAMP binds to CAP, the complex binds to the promoter region of the genes that are needed to use the alternate sugar sources. In these operons, a CAP-binding site is located upstream of the RNA-polymerase-binding site in the promoter. This increases the binding ability of RNA polymerase to the promoter region and the transcription of the genes. As cAMP-CAP is required for transcription of the lac operon, this requirement reflects the greater simplicity with which glucose may be metabolized in comparison to lactose.
The Initiation Complex and Translation Rate
The first step of translation is ribosome assembly, which requires initiation factors.
Discuss how eukaryotes assemble ribosomes on the mRNA to begin translation
- The components involved in ribosome assembly are brought together by the help of proteins called initiation factors which bind to the small ribosomal subunit.
- Initiator tRNA is used to locate the start codon AUG (the amino acid methionine) which establishes the reading frame for the mRNA strand.
- GTP carried by eIF2 is the energy source used for loading the initiator tRNA carried by the small ribosomal subunit on the correct start codon in the mRNA.
- GTP carried by eIF5 is the energy source for assembling the large and small ribosomal subunits together.
- reading frame: either of three possible triplets of codons in which a DNA sequence could be transcribed
- phosphorylation: the addition of a phosphate group to a compound; often catalyzed by enzymes
Ribosome Assembly and Translation Rate
Like transcription, translation is controlled by proteins that bind and initiate the process. In translation, before protein synthesis can begin, ribosome assembly has to be completed. This is a multi-step process.
In ribosome assembly, the large and small ribosomal subunits and an initiator tRNA (tRNAi) containing the first amino acid of the final polypeptide chain all come together at the translation start codon on an mRNA to allow translation to begin. First, the small ribosomal subunit binds to the tRNAi which carries methionine in eukaryotes and archaea and carries N-formyl-methionine in bacteria. (Because the tRNAi is carrying an amino acid, it is said to be charged.) Next, the small ribosomal subunit with the charged tRNAi still bound scans along the mRNA strand until it reaches the start codon AUG, which indicates where translation will begin. The start codon also establishes the reading frame for the mRNA strand, which is crucial to synthesizing the correct sequence of amino acids. A shift in the reading frame results in mistranslation of the mRNA. The anticodon on the tRNAi then binds to the start codon via basepairing. The complex consisting of mRNA, charged tRNAi, and the small ribosomal subunit attaches to the large ribosomal subunit, which completes ribosome assembly. These components are brought together by the help of proteins called initiation factors which bind to the small ribosomal subunit during initiation and are found in all three domains of life. In addition, the cell spends GTP energy to help form the initiation complex. Once ribosome assembly is complete, the charged tRNAi is positioned in the P site of the ribosome and the empty A site is ready for the next aminoacyl-tRNA. The polypeptide synthesis begins and always proceeds from the N-terminus to the C-terminus, called the N-to-C direction.
In eukaryotes, several eukaryotic initiation factor proteins (eIFs) assist in ribosome assembly. The eukaryotic initiation factor-2 (eIF-2) is active when it binds to guanosine triphosphate (GTP). With GTP bound to it, eIF-2 protein binds to the small 40S ribosomal subunit. Next, the initiatior tRNA charged with methionine (Met-tRNAi) associates with the GTP-eIF-2/40S ribosome complex, and once all these components are bound to each other, they are collectively called the 43S complex.
Eukaryotic initiation factors eIF1, eIF3, eIF4, and eIF5 help bring the 43S complex to the 5′-m7G cap of an mRNA be translated. Once bound to the mRNA’s 5′ m7G cap, the 43S complex starts travelling down the mRNA until it reaches the initiation AUG codon at the start of the mRNA’s reading frame. Sequences around the AUG may help ensure the correct AUG is used as the initiation codon in the mRNA.
Once the 43S complex is at the initiation AUG, the tRNAi-Met is positioned over the AUG. The anticodon on tRNAi-Met basepairs with the AUG codon. At this point, the GTP bound to eIF2 in the 43S complexx is hydrolyzed to GDP + phosphate, and energy is released. This energy is used to release the eIF2 (with GDP bound to it) from the 43S complex, leaving the 40S ribosomal subunit and the tRNAi-Met at the translation start site of the mRNA.
Next, eIF5 with GTP bound binds to the 40S ribosomal subunit complexed to the mRNA and the tRNAi-Met. The eIF5-GTP allows the 60S large ribosomal subunit to bind. Once the 60S ribosomal subunit arrives, eIF5 hydrolyzes its bound GTP to GDP + phosphate, and energy is released. This energy powers assembly of the two ribosomal subunits into the intact 80S ribosome, with tRNAi-Met in its P site while also basepaired to the initiation AUG codon on the mRNA. Translation is ready to begin.
The binding of eIF-2 to the 40S ribosomal subunit is controlled by phosphorylation. If eIF-2 is phosphorylated, it undergoes a conformational change and cannot bind to GTP. Therefore, the 43S complex cannot form properly and translation is impeded. When eIF-2 remains unphosphorylated, it binds the 40S ribosomal subunit and actively translates the protein.
The ability to fully assemble the ribosome directly affects the rate at which translation occurs. But protein synthesis is regulated at various other levels as well, including mRNA synthesis, tRNA synthesis, rRNA synthesis, and eukaryotic initiation factor synthesis. Alteration in any of these components affects the rate at which translation can occur.