MCB 201 Gene Expression - Spring Semester 2003
Lecture 14 (Regulation of Transcription Initiation cont.)
1. Figure 10-28. Approximate mapping of transcription-initiation site by analysis of nascent transcripts synthesized in vivo. The key to this approach is the production of RNA during a short radioisotopic labeling period. Then the cells are disrupted and the RNA is separated by velocity sedimentation centrifugation, roughly by size. Each fraction collected from the velocity gradient is hybridized to restriction fragments from the gene of interest. The shortest RNA transcripts will hybridize to the DNA fragment containing the start site for transcription (fragment B). The largest RNA molecules will bind to DNA fragment D containing the start site for transcription. Note that the bottom part of this figure does not clearly show that the size-selected mRNAs are added to test tubes containing only one class of DNA fragment for hybridization analysis.
2. Figure 10-29. Precise mapping of initiation site of adenovirus late
transcription unit by in vitro transcription. This figure shows how one
of the first start sites was precisely mapped. This approach uses DNA fragments
from the region of the start site to program cell-free transcription assays
and radioactive RNA is synthesized. The RNA molecules are capped on their
5' ends, as discussed below, and the nucleotide attached to the cap can
be identified. Transcription stops when the RNA polymerase comes to the
end of the DNA template and falls off. These radioactive RNA transcripts
were then separated by gel electrophoresis (Figure 10-29 right). The positions
of the restriction sites in the DNA template are known, so the lengths of
the RNA transcripts in nucleotides precisely map the start site at 16.4
map units on the adenovirus genome. The diagram shows that this sequence
corresponds exactly to the 5' end of authentic, capped mRNA.
Current practice usually involves identifying the DNA sequence corresponding
to the 5' end of the mRNA using primer extension and nuclease protection
assays. This then is the transcription start site.
3. Figure 7-35. Two methods for mapping the start site for transcription of a particular gene in a region of DNA of known sequence. Both methods start with the mRNA corresponding to a gene and a piece of single-stranded DNA labeled at the 5' end that is complementary to the mRNA. in the case of S1 nuclease mapping, shown in Panel A, this piece of DNA, called an S1 probe, is hybridized to the mRNA. Then this product is digested with S1 endonuclease, which only cuts single-stranded nucleic acids (RNA and DNA). The resulting double-stranded RNA-DNA hybrid is denatured and the radioactively labeled, single-stranded DNA is sized by electrophoresis. The length of this fragment is the distance between the starting or first nucleotide at the 5' end of the mRNA and the labeled 5' end of the S1 DNA probe. In part C, another method called primer extension is used. Here a small single-stranded DNA primer labeled on its 5' end is hybridized to the mRNA. This serves as a primer for reverse transcriptase, which used the RNA as a template to extend the length of the DNA primer to the 5' end of the mRNA molecule. This product is denatured and the radioactive single-stranded DNA molecule is sized by gel electrophoresis. The length of the primer extension product measures the distance from the 5' end of the primer to the 5' end of the mRNA.
Section 10.4 (Regulatory Sequences in Eukaryotic Protein-Coding Genes)
4. Introduction. This section is about the various kinds of elements found in the transcriptional-control regions of eukaryotic protein-coding genes. They are summarized here:
5. Figure 10-30. Comparison of nucleotide sequences upstream of the start site in 60 different vertebrate protein-coding genes. The diagram shows how a 'consensus' sequence is identified. These sequences are located about 25-35 base pairs upstream of the start site for transcription. RNA polymerase II is particularly dependent on the TATA box for initiation. It appears to physically position the polymerase to start initiation in the correct place, since shortening the number of nucleotides between the TATA box and original start site shifts the start site to maintain the 25-35 base pair distance.
6. Some eukaryotic genes contain an 'initiator' element instead of a TATA box. Unlike the TATA box with its clear consensus sequence, initiator sequences are extremely degenerate. However like the TATA box, the initiator element positions polymerases to initiate transcription at a well-defined site.
(5') YYA+1N-T/A-YYY (3')
Y= C or T (pyrimidines)
N= any one of the 4 bases
A+1= start site of transcription
7. CpG islands. These are CG (cytosine and guanine) rich sequences of usually 20-50 nucleotides positioned about 100 base pairs upstream from the start site. They are susceptible to being cut by restriction enzymes like HapII that include CG in their recognition sequences. The characteristic of this type of promoter region is that initiation does not start at only one site, but rather can start at different sites within a 20-200 base pair range. The products are RNAs with different 5' ends and 5' untranslated regions, but the same coding sequence. Interestingly, the C before the G is a relatively frequent site of DNA methylation, which reduces transcriptional activity.
8. Figure 10-31. Analysis of linker scanning mutations to identify transcription control elements. The Lodish text uses the term 'promoter-proximal elements' for transcriptional control elements not including the promoter elements described above that lie within 100-200 base pairs upstream of the start site. The approach to identifying these regions, shown in this figure, actually first requires the production of a set of 5' deletions, as shown in Figure 10-24. Once the 5' border of a transcription control region has been found, then the sequence can be carefully mapped using linker scanning mutations, as described in figure 10-31. Note that in this example, reporter gene expression is used to assay for the effect of the mutation. Where the linker knocks down expression is the presumed site of a control element. Note that a linker is a small DNA segment, about 10 bases, with a scrambled coding sequence, so it acts like an insertional mutation.
9. Figure 10-32 (A, B, C). Identification of promoter-proximal elements controlling the thymidine kinase (tk) gene of herpes simplex virus (HSV) by analysis of linker scanning (LS) mutations. In this specific example of a search strategy for these control elements, gene activity is assayed by measuring the production of RNA. This is done by using the primer-extension method. A small radioactively labeled DNA primer is hybridized to the RNA product and then this primer is extended by reverse transcriptase to make a relatively short DNA product. This DNA is then separated by gel electrophoresis. Note that the assay results in two DNA products, one slightly shorter than the other, presumeably an artefact of the reverse transcriptase reaction. Note that the pseudo wild-type construct (called pseudo because a piece was deleted from the coding region) is injected into the same oocyte as the LS (Linker Scanner) mutant DNA to serve as a positive control for oocyte transcription and recovery of the RNA product. The deleted region is between the primer binding site and the end of the RNA transcript, resulting in a shorter extension product that can be distinguished on gels from the primer extension product from the LS mutant DNA. Note that the DNA corresponding to the RNA from this control construct is fairly uniform across the gel, whereas the DNA made from the LS RNA varies a lot depending upon whether the linker is sitting in a control element or not.