Cellular and Molecular Biology Topics
RNA Synthesis
RNA synthesis is similar to DNA synthesis in that it requires a template and the new chain grows 5 to 3 by addition of nucleotides at the 3 end. Both use similar activated precursors (5 rNTPs for RNA), are catalyzsed by a polymerase (RNA polymerase) and the synthesis reaction have similar chemistry (3OH attach of the innermost a-phosphate, displacing pyrophoasphate).
RNA synthesis is different from DNA synthesis in that a primer is not needed, only one strand is transcribed and the parental double helix reforms.
In bacteria, a single transcription unit (an operon) can contain several genes. An operon is the unit of gene expression, including regulatory and coding elements. Often a cluster of geners is regulated as a single unit. An operator is a short DNA sequence that binds a repressor protein and controls transcription of the adjacent gene(s).
Transcription beguins at a specific base pair determined by a DNA sequence called a promoter, which is a binding site for RNA polymerase. Polymerase first binds to form a closed complex in which the template DNA strands are not yet separated. Then it separates the strands over a distance of 12-13 base pairs surrounding the transcription start site. This forms an open complex (a "bubble") and polymerization of the RNA strand complementary to the coding strand starts.
The transcription "bubble" moves along, with DNA opening in front and closing behind. A short stretch of RNA:DNA hybrid is present, but the transcript is eventually displaced and the parental strands rewind as polymerase moves along.
Termination of transcription can be dependent on the rho factor or be rho-independent. In rho-dependent termination, rho binds to RNA and moves along it using ATP. When it catches up with polymerase, it triggers the enzyme to stop synthesis and dissociate from the coding strand. Rho-independent termination occurs by the synthesis of a self-complimentary sequence that base-pairs with itself forming a loop. This signals polymerase to release the template.
E. coli polymerase is composed of several subunits: a2bbs. The core enzyme that catalyzes transcription is the a2bb complex, while s is an initiation factor, released from the catalytic core after initiation. RNA polymerase is responsible for initiation of transcription, RNA synthesis and termination of transcription.
Take Quiz: [Q1] [Q2] [Q3]
Advance Topics: Medical Pharmacology
Terminology
A promoter is a DNA sequence that allows RNA polymerase to bind and start transcription.
In prokaryotes, the sequence of a promoter is recognized by the Sigma (s)
factor of the RNA polymerase. In eukaryotes, it is recognized by specific transcription
factors.
An E. coli promoter has a sequence called a Pribnow box, which is on average TATAAT, about 10 nucleotides upstream from were the RNA polymerase will start synthesis. There is another promoter box 30-35 nucleotides upstream with the average sequence TTGACA. Genes whose promoters are very similar to these average sequences are transcribed at a higher rate because they are good substrates for the a2bbs RNA polymerase (i.e. with the s subunit).
Inducers are small molecules that induce the expression of an operon. A repressor is a protein that represses expression of an operon. Aporepressors bind to operators only if bound to a specific ligand. Co-repressor is the ligand that binds to and activates an aporepressor.
Catabolite repression occurs when a break down product of metabolism prevents synthesis of mRNA encoding specific proteins. Cyclic adenosine-35-monophosphate (cAMP) is a common catabolite and intracellular signal that often acts as a co-repressor or co-promoter.
A constitutive gene is expressed steadily (in other words, is always on), although the amount of transcription is still controlled by the promoter. A repressible gene is expressed unless a repressor is present. An inducible gene is not expressed unless an inducer is present.
Monocistronic mRNA encodes only one polypeptide chain. Polycistronic mRNA encodes
multiple peptide chains, each independently initiated from a particular sequence.
About one fourth of E. coli mRNA is polycistronic. Eukaryotes have
mostly monocystronic mRNA.
A regulon is a group of operons under common control, i.e. they have a common repressor. Damage to E. coli DNA triggers a coordinated response called the SOS response. Which is regulated by lexA. The SOS regulon includes the lexA gene along with many other genes encoding enzymes that act to repair damage. In undamaged cells, most of the SOS genes are largely repressed by binding of lexA to the operator site in the regulatory region. When damage blocks replication forks leaving single stranded DNA, RecA binds and is activated, causing the cleavage of lexA. The cleaved lexA cannot bind to operators, so the set of operons in the SOS regulon is induced. When damage is repaired, RecA is deactivated and no longer cleaves lexA.
Take Quiz: [Q1] [Q2] [Q3]
Back to Basics: Biochemistry
The lac Operon
Three important operons in E. coli are the lac operon and the trp operon the SOS regulon. The lac operon is a classic example of a catabolite-regulated operon, while the trp operon is an example of an attenuated operon.
The lac operon includes regulatory elements and genes that transcribe three polypeptides needed for lactose metabolism: b-galactosidase (z), permease (y) and transacetylase (a). The regulatory element includes a promoter sequence (P) and an operator (O). In the absence of lactose, a repressor binds to the operator in the lac operon. This repressor is coded by the lac I gene (i), which happens to be just upstream of the lac operon (this is not necessarily the case for other repressor genes).
The normal or ground state of an E. coli cell is to have the lac operon repressed because there is enough glucose in the environment, which is preferentially used for energy instead of lactose.When lactose and glucose are both available a modified lactose called allolactose binds the repressor, changing its shape in a way that it can no longer bind to the operator.
With the operator free, RNA polymerase can bind to and transcribe DNA. But the prescense of glucose has a dampening effect on transcription of the lac operon by a mechanism involving cAMP. Cyclinc AMP activates a protein called catabolite activation protein (CAP) which binds to the lac operon promoter and accelerates transcription. But in the presence of glucose, there is little cAMP available and the lac operon is transcribed at a lower rate. When glucose is not available, cAMP levels are high and available to bind and activate CAP, which in turn binds to the lac operon promoter and accelerates transcription.
Take Quiz: [Q1] [Q2] [Q3]
Back to Basics: Biochemistry
The trp Operon
The trp operon encodes the genes for the synthesis of tryptophan (Trp). When Trp levels are high, it acts as a co-repressor and binds to an aporepresor. The trp aporepressor binds to the trp operator sequence. When Trp levels are low, the aporepressor cannot bind to the operator of the Trp operon and transcription occurs at a high rate.
Expression of the trp operon is also regulated by attenuation. The attenuator region is a sequence found within the transcribed RNA, near the 5' end and before the first trp gene. This transcribed sequence, also known as the leader sequence, controls transcription from the mRNA.
The attenuator sequence contains tandem repeats of the tryptophan codon and is capable of forming several different stable stem-loop structures. Because of the tandem Trp codons and depending on the level of tryptophan in the cell, the rate of translation allows different stem-loops to form. If tryptophan is abundant, the ribosome favors formation of a loop near a region rich in uracil. Such a loop acts as the transcriptional terminator loop, therefore RNA polymerase is dislodged from the template.
When tryptophan is low, a ribosome translating the leader arrives at the two tryptophan codons and must wait for a tRNA carrying tryptophan. During this pause, the RNA polymerase continues transcribing, and the pause allows the formation of a stem loop forms away from the uracil-rich region. Therefore, translation is not terminated and will continue after two Trp tRNAs reach the ribosome.
When the leader peptide is being translated under high tryptophan conditions, the ribosome does not pause at the two Trp codons. In this case, a stem loop forms near an uracil-rich region, signaling termination of transcription.
Take Quiz: [Q1] [Q2] [Q3]
Back to Basics: Biochemistry
Eukaryotic RNA Polymerases
Eukaryotes have three types of RNA polymerase: RNA polymerases II, II and III. RNA polymerase I synthesizes most ribosomal RNA (rRNA), except 5s. RNA polymerase II synthesizes all mRNA. RNA Polymerase III synthesizes transfer RNA (tRNA) and 5s rRNA.
During RNA synthesis, the first nucleotide is added at the +1 or cap site. Each type of polymerase responds to a different set of promoters surrounding the cap site. The promoter regions of RNA polymerase I and III are much more simple than that of RNA polymerase II.
There are two critical sequences in the promoter region of RNA polymerase I: one surrounds the capsite (proximal domain), extending from about 40 to +5, the other is about 100 base pairs upstream (upstream control element or UCE).
Both sites are bound by the same transcription factor, the Upstream Binding Factor (UBF), which associates with the protein SL1. SL1 is a complex of TATA binding protein, even though these promoters do not have a TATA binding box (a promoter for RNA polymerase II).
The critical cis-acting elements for RNA Polymerase III are within the region that is being transcribed. For example, in the 5s rRNA gene there is a key sequence from +55 to +80 to which a key transcription factor called Transcription Factor for Polymerase III (TFIIIA) binds.
There is a TFIIIC that binds to TFIIIA, and a TFIIIB that binds around the cap site. This complex stays bound even while polymerase transcribes pass it. The complex is bound with nine zinc fingers and is thought that several of the fingers can release while the polymerase synthesizes by them, and bind back after polymerase has passed.
Take Quiz: [Q1] [Q2] [Q3]
Advance Topics: Molecular Biology Lab
Polymerase II Promoters
The typical RNA polymerase II gene promoters are expressed in tissue specific patterns and contain an array of cis-acting elements. These differ in type, sequence and position in different genes. Two important promoters that define the cap site are the TATA box and the initiator. Some genes have both a TATA box and initiator, others have one or the other.
The TATA box is usually 20-30 nucleotides upstream from the cap site. Its consensus sequence, TATAAA, is quite similar to the -10 region of the prokaryotic s recognition site.The initiator has a more complicated sequence and flanks the cap site. TATA binding proteins (TBP) bind to either the TATA box or the initiator or both, signaling the start point of transcription.
The TATA box and initiator are known as proximal elements, as opposed to upstream elements which are at a longer, variable distance from the cap site. Other proximal elements sometimes present upstream from the TATA box are G-C box, CCAAT box and tissue-specific sequnces.
The G-C box and CCAAT box can bind with a protein called Sp1 and other regulatory proteins. These interactions stimulate transcription from the start site defined by the TATA and initiator sequence. Tissue-specific sequences can be found scattered upstream of the G-C or CCAAT boxes. How strongly these sequences match a consensus defines how strongly the genes is transcribed.
At a variable distance from the G-C or CCAAT boxes are upstream cis-acting elements called enhancers, which typically bind a complex of proteins that stimulate transcription, and receptor binding sites (I am not sure, are receptor binding sites enhancers or are they two different things?). Unlike other promoters, enhancers do not direct were the transcription occurs or in what direction, but enhance transcription from a nearby promoter.
Cells
differ in the transcription factors they express and in the concentrations of
each transcription factor. Therefore, different complexes will assemble at a
promoter in different cells. Not all sites may be occupied in a given tissue,
allowing many possible combinations. Usually, the more cis-element sites are
occupied by trans-elements, the higher the expression of that particular gene.
Take Quiz: [Q1] [Q2] [Q3]
Advance Topics: Medical Pharmacology
Topic
Incomplete.
Take Quiz: [Q1] [Q2] [Q3]
Advance Topics: Molecular Biology Lab
Topic
Incomplete.
Take Quiz: [Q1] [Q2] [Q3]
Advance Topics: Molecular Biology Lab
Topic
Incomplete.
Take Quiz: [Q1] [Q2] [Q3]
Advance Topics: Molecular Biology Lab
Topic
Incomplete.
Take Quiz: [Q1] [Q2] [Q3]
Advance Topics: Molecular Biology Lab
Topic
Incomplete.
Take Quiz: [Q1] [Q2] [Q3]
Advance Topics: Molecular Biology Lab
Topic
Incomplete.
Take Quiz: [Q1] [Q2] [Q3]
Advance Topics: Molecular Biology Lab
Continue
to "Eukaryotic Gene Expression" or take a test: [T1] [T2] [T3].
Need more practice? Answer the following review questions:
Questions not yet available