MCB 201 Gene Expression - Spring Semester 2003
Lecture 19 (chapter 11 RNA Processing, Nuclear Transport, and Post-transcriptional Control)
Section 11.1: Transcription Termination
1. Whereas, initiation of transcription is the most frequent step at which genes are regulated, there are a number of possible control points after this step. i.e. downstream from initiation, and all of them are used somewhere in organisms. All of these regulatory mechanisms fall into the category of posttranscriptional control.
2. We will start with transcription termination as our first downstream mechanism, and we will use an example from bacteria, where this process is best understood. The most frequently used mechanism is called Rho-independent termination, and this is where we will concentrate. There is another mechanism, Rho-dependent termination, which uses a protein called Rho as a transcription termination factor.
3. Figure 11.1: Sequence of trp termination (t) site, a Rho-independent site. These DNA sites have two characteristic features: 1) A DNA segment that encodes a GC-rich complementary base-paired region of transcribed RNA followed by 2) DNA segment that contains a series of A residues that are transcribed into a series of U residues in the RNA transcript (poly U region). Note that both of these characteristic features are encoded in the termination site DNA, but we recognize their consequences in the RNA transcript. The stem-loop structure that is formed by the GC base pairs n the RNA transcript is thought to bind to the E. coli RNA polymerase and cause it to pause during transcription. In addition the AU base pairs that form in the DNA:RNA hybrid during transcription are weaker than regions containing GC pairs, and this encourages release of the RNA chain from the transcription complex.
4. Figure 11.2: Attenuation provides a secondary mechanism for controlling expression of the trp operon. Recall that this operon encodes the genetic information to make the biosynthetic enzymes that produce the amino acid tryptophan in bacterial cells. The leader sequence (L) in the DNA just preceding the first structural gene (E) contains an attenuator site (marked by red band). In cells with high trp levels, a short piece of RNA is transcribed, i.e. transcription is attenuated. In cells with low trp levels, the complete mRNA is made.
5. Figure 11.3: Mechanism of attenuation of trp-operon transcription. (A) Here is a more detailed diagram of the trp leader RNA transcribed from the trp DNA. Four regions are shown in color that can form alternate stem-loop structures, but only one of these, the 3-4 stem-loop leads to attenuation. To understand the attenuation mechanism, recall that in bacteria, translation can start at the 5' end of mRNA even before it is finished being transcribed. In Panel B, a ribosome has bound to the mRNA. When tryptophan levels are high in the cell, the ribosome moves quickly through region 1 and into region 2, covering it so that it cannot participate in forming a 2-3 stem-loop structure This allows time for stem-loop 3-4 to form, which is followed by a poly U region, the two characteristics needed for attenuation. When this structure forms, RNA polymerase terminates transcription, suggesting that the polymerase somehow interacts with this stem-loop. Under conditions of low tryptophan in cells, this operon needs to be turned on, i.e. transcribed fully. How does the cell 'sense' the tryptophan concentration and communicate this to the attenuator? Region 1 of the leader RNA contains two successive tryptophan codons. When trp levels are low, the ribosome pauses at each of these codons, waiting for a tRNA charged with trp, but because trp levels are low in the cell, the levels of tRNA-trp are low and the ribosome has to wait. This allows segment 2 time to form a stem-loop with segment 3, and stem-loop 2-3 is not a terminator because it lacks the poly U sequence after it. This allows RNA polymerase to continue transcribing. Remember that segment 3 can pair with either segment 2 or 4, with very different consequences.
6. How do eukaryotic RNA polymerases terminate? Each type of polymerase uses a different mechanism. Pol I, which transcribes rRNA genes, uses a DNA-binding protein that binds downstream of the transcription unit. Pol III, which transcribes tRNA genes, terminates after transcribing a run of U residues. No stem-loops are required as in bacterial termination. Pol II, which transcribes protein-coding genes, uses a termination mechanism that is coupled to the cleavage and polyadenylation of the 3' ends of the pre-mRNA. In yeast, the cleavage event appears to be the essential event signalling termination. The complex of enzymes that carries out these reactions binds to the phosphorylated CTD domain of pol II and this may help to coordinate cleavage, termination and polyadenylation.
7. Figure 11.6: Here is a eukaryotic example of a control mechanism that involves a choice between chain elongation or termination. The transcription of the human immunodeficiency virus (HIV) genome is regulated by an antitermination mechanism. Two proteins, Tat and cyclin T bind to a sequence called TAR (a stem-loop structure) near the 5' end of the HIV RNA transcript. These two proteins help position another protein, Cdk9, a protein kinase that phosphorylates the CTD of Pol II. This prevents termination and allows Pol II to continue transcription.
11.2 Processing of Eukaryotic mRNA
1. Figure 11-7: Overview of mRNA processing in eukaryotes (media connections: Life Cycle of an mRNA). Eukaryotic mRNA processing involves three major processes: a) 5' capping, b) 3' cleavage/polyadenylation, and c) RNA splicing. Note that capping occurs shortly after transcription begins, and that for large genes with many introns, splicing may begin before transcription is terminated.
2. This is a good point at which to introduce two figures taken from the review article handed out in class by Orphanides and Reinberg entitled "Unified Theory of Gene Expression". In these figures, the traditional view of gene expression is compared with a contemporary view:
Figure A: A traditional view of gene expression. The different steps in the pathway from gene to protein have traditionally been viewed as independent events, with each going to completion before the next begins. (figure legend of Figure 1 from review article).
Figure B: A contemporary view of gene
expression. Recent findings suggest that each step regulating gene expression
(from transcription to translation) is a subdivision of a continuous process.
In this contemporary view of gene expression, each stage is physically and
functionally connected to the next, ensuring that there is efficient transfer
between manipulations and that no individual step is omitted. (figure legend
of Figure 2 from review article).
3. Figure 11-8. We have already talked about capping of the 5' end of nascent RNA transcripts with 7'-methylguanylate. We did not mention earlier, that the capping enzyme associates with the phosphorylated CTD end of Pol II, so this is a very busy interaction domain (see Figure B above).
4. Figure 11-9: Visualization of hnRNP protein associated with the nascent transcripts in an oocyte (egg cell) of a newt . The white staining of the DNA in the chromosome axis is due to a stain called DAPI.The long red filaments are nascent RNA transcripts coated with hnRNP proteins that have been bound with a monoclonal antibody tagged with rhodamine, which fluoresces red under ultraviolet light. hn = heterogeneous nuclear, and recalls one of the first properties found for nuclear RNA primary transcripts, i.e. they are very heterogeneous in size. In fact, pre-mRNA molecules do not exist in cells as free RNA. They are always bound to proteins to form ribonucleoprotein complexes or RNPs.
5. Figure 11-10: A structural motif. Structure of complex between an RNP motif from U1A protein and RNA. Panel A: The evolutionarily conserved RNP1 and RNP2 regions are located in the two middle beta-strands. Panel B: Note that the RNA forms a stem-loop, with the single-stranded part of the loop bound to the surface of the protein.
6. Figure 11-11: Hybridization of RNA molecules in vitro is accelerated by hnRNP proteins. In the absence of proteins, the secondary structure in RNA slows hybridization between complementary chains. Proteins bound to the RNA prevent the formation of these RNA secondary structures, thus promoting hybridization between RNA molecules. Intermolecular hybridization is important for RNA splicing, for example. hnRNP proteins may assist in processing and transport of mRNAs, again by removing RNA secondary structure and facilitating the interaction of the RNA with other molecules, perhaps translocation components for example.
