2 a. RNA polymerase II that is actively transcribing a gene (actively progressing along the DNA polymerizing an RNA molecule) is phosphorylated. Researchers evaluated the abundance (the frequency of occupancy) of RNA PolII at difference positions along a gene, and correlated that with the introns and exons of the gene. The figure represents a portion of one gene that is transcribed from left to right. The X axis corresponds to position along the DNA, and the boxes below the figure illustrate where the exons are (with the lines between them representing the introns). The Y axis represents the relative frequency of occupancy (relative abundance of active RNA PolII using two different measures [pSer2 and pSer5]). A high the value on this axis means that, when they sample whether RNA polII is present at the given position, they find the enzyme present more frequently than at positions where the value is low. a. With respect to intron/exon boundaries, what positions does the (transcribing) RNA PolII occupy most? b. How do you interpret this observation (i.e., does it say anything about how fast or how slow RNA PolII is moving at different positions across the gene)? Does it suggest anything about the relationship between transcription and splicing? 2b. You characterize the sequence of a full-length cDNA and the corresponding genomic DNA for a particular intron-containing gene from mouse cells. When you align them to each other using a computer program, the exons of the cDNA align perfectly in some regions with pieces of the genomic DNA, whereas other exons appear to have a small number of specific nucleotide differences compared to the genomic DNA. Assume this genomic DNA and cDNA come from the same individual, and there are no sequencing mistakes. What might account for these differences? ,241 132 15 98 459605 719M850 1341/1417 Ratio pser relative to RNAPI Solution 2a RNA polymerase II at first recognizes and binds to the promoter DNA forming a state termed as the closed complex. Following this the DNA surrounding the transcription start site is unwound and the template strand is positioned in the Polymerase active site, forming the open complex. Transcription initiation then commences, initially producing short RNA products. b Alternative splicing is a crucial mechanism for gene regulation and for generating genomic diversity. Recent studies indicate that the expression of nearly 95% of human multi-exon genes involves alternative splicing. In metazoans, alternative splicing plays an important role in generating different protein products that function in diverse cellular processes including cell growth, differentiation and death. Splicing is carried out by the spliceosome. A massive structure in which five small nuclear ribonucleoprotein particles (snRNPs) and a large number of auxiliary proteins cooperate to accurately recognize the splice sites and catalyse the two steps of the splicing reaction. 2b There is consistent evidence that both proce.

1. 2 a. RNA polymerase II that is actively transcribing a gene (actively progressing along the DNA
polymerizing an RNA molecule) is phosphorylated. Researchers evaluated the abundance (the
frequency of occupancy) of RNA PolII at difference positions along a gene, and correlated that
with the introns and exons of the gene. The figure represents a portion of one gene that is
transcribed from left to right. The X axis corresponds to position along the DNA, and the boxes
below the figure illustrate where the exons are (with the lines between them representing the
introns). The Y axis represents the relative frequency of occupancy (relative abundance of active
RNA PolII using two different measures [pSer2 and pSer5]). A high the value on this axis means
that, when they sample whether RNA polII is present at the given position, they find the enzyme
present more frequently than at positions where the value is low.
a. With respect to intron/exon boundaries, what positions does the (transcribing) RNA PolII
occupy most?
b. How do you interpret this observation (i.e., does it say anything about how fast or how slow
RNA PolII is moving at different positions across the gene)? Does it suggest anything about the
relationship between transcription and splicing?
2b. You characterize the sequence of a full-length cDNA and the corresponding genomic DNA
for a particular intron-containing gene from mouse cells. When you align them to each other
using a computer program, the exons of the cDNA align perfectly in some regions with pieces of
the genomic DNA, whereas other exons appear to have a small number of specific nucleotide
differences compared to the genomic DNA. Assume this genomic DNA and cDNA come from
the same individual, and there are no sequencing mistakes. What might account for these
differences? ,241 132 15 98 459605 719M850 1341/1417 Ratio pser relative to RNAPI
Solution
2a RNA polymerase II at first recognizes and binds to the promoter DNA forming a state termed
as the closed complex. Following this the DNA surrounding the transcription start site is
unwound and the template strand is positioned in the Polymerase active site, forming the open
complex. Transcription initiation then commences, initially producing short RNA products.
b Alternative splicing is a crucial mechanism for gene regulation and for generating genomic
diversity. Recent studies indicate that the expression of nearly 95% of human multi-exon genes
involves alternative splicing. In metazoans, alternative splicing plays an important role in
generating different protein products that function in diverse cellular processes including cell
growth, differentiation and death.
Splicing is carried out by the spliceosome. A massive structure in which five small nuclear

2. ribonucleoprotein particles (snRNPs) and a large number of auxiliary proteins cooperate to
accurately recognize the splice sites and catalyse the two steps of the splicing reaction.
2b There is consistent evidence that both processes are coordinated and in some cases
functionally coupled. Studies have been done on the roles of cis- and trans-acting factors that
regulate transcription, on constitutive and alternative splicing. Recent evidence suggests that
transcriptional elongation and splicing can be influenced reciprocally i.e, elongation rates control
alternative splicing and splicing factors in turn modulate polymerase II elongation. The presence
of transcription factors in the spliceosome and the existence of proteins such as the coactivator
PGC-1 with dual activities in splicing and transcription can explain the link between both
processes and add a new level of complexity to the regulation of gene expression in eukaryotes.