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u The DNA control elements in eukaryotic genomes that bind transcription factor often are located much farther from the promoter they regulate than is the case in prokaryotic genomes. u In some cases, transcription factors that regulate expression of protein-coding genes in higher eukaryotes bind at regulatory sites tens of thousands of
base pairs either upstream or downstream from the promoter. u Transcription of a single gene may be regulated by binding of multiple transcription factors to alternative control elements, directing expression of the same gene in different types of cells and at different times during development.
u Several transcription-control DNA sequences regulate expression of the mammalian gene encoding the transcription factor Pax 6. u Heterozygous humans with only one functional Pax 6 gene are born with aniridia, a lack of irises in the eyes. u The Pax 6 gene is expressed from at least three alternative promoters that
function in different cell types and at different times during embryogenesis. u Analysis of transgenic mice with smaller fragments of DNA from this region allowed the mapping of separate transcription-control regions regulating transcription in the pancreas and in the lens and cornea.
u Drosophila contains three RNA polymerase I, II, and III. u Three enzymes exhibit various net charges. u RNA polymerase I is very insensitive to α-amanitin, but RNA polymerase II is very sensitive-the drug binds near the active site of the enzyme; RNA polymerase III is intermediate.
u Each eukaryotic RNA polymerase catalyzes transcription of genes encoding different classes of RNA. u RNA polymerase I, located in the nucleolus, transcribes genes encoding precursor r. RNA (pre-r. RNA), which is processed into 28 S, 5. 8 S, and 18 S r. RNA. u RNA polymerase III transcribes genes encoding t. RNAs, 5 S r. RNA, and small
, stable RNAs, including one involved in RNA splicing (U 6) and the RNA component of the signal-recognition particle (SRP) involved in directing nascent proteins to the endoplasmic reticulum. u RNA polymerase II transcribes all protein-coding genes; it functions in production of m. RNAs. RNA polymerase
II produces four of the five small nuclear RNAs that take part in RNA splicing. u RNA polymerase II transcribes all protein-coding genes; it functions in production of m. RNAs. RNA polymerase II also produces four of the five small nuclear RNAs that take part in RNA splicing.
u Each of the three eukaryotic RNA polymerase is more complex than E. coli RNA polymerase with similar structures.
u All three contain two large subunits and 10 -14 smaller subunits. u The two large subunits (RPB 1 and RPB 2) of all three eukaryotic RNA polymerases are similar to the E. coli β’ and β subunits. Each of the eukaryotic polymerases also contains a ω-like and two α-like subunits.
u In addition to their core subunits related to the E. coli RNA polymerase subunits, all three yeast RNA polymerases contain four additional small subunits, common to them but not to the bacterial RNA polymerase.
u All subunits are necessary for eukaryotic RNA polymerase to function normally. u The carboxyl end of the largest subunit of RNA polymerase II (RPB 1) contains a stretch of seven amino acids that is nearly precisely repeated multiple times.
u This heptapeptide repeat, with a consensus sequence of Tyr-Ser-Pro-Thr. Ser-Pro-Ser is known as the carboxylterminal domain (CTD). u The CTD is critical for viability. u RNA polymerase II molecules that initiate transcription have a unphosphorylated CTD.
u Once the polymerase initiates transcription and begins to move away from the promoter, many of the serine and some tyrosine residues in the CTD are phosphorylated. u The large chromosomal puffs induced at this time in development
are regions where the genome is very actively transcribed. u The RNA polymerase II associated with the highly transcribed puffed regions contains a phosphorylated CTD.
are regions where the genome is very actively transcribed. u The RNA polymerase II associated with the highly transcribed puffed regions contains a phosphorylated CTD. u The transcripts produced always contained a cap structure at their 5’ end; the 5’ cap is added to the nascent RNA which by enzymes in the nucleus.
u In all transcribed genes, a conserved TATA box was found 2535 base pairs upstream of the start site. u TATA box acts similarly to an E. coli promoter to position RNA polymerase II for transcription initiation.
u Instead of a TATA box, some eukaryotic genes contain an alternative promoter element called an initiator. u Most naturally occurring initiator elements have a cytosine (c) at the -1 position and an adenine (A) residue at the transcription start site (+1).
u The nucleotide sequence immediately surrounding the start site determines the strength of such promoters. u Only an extremely degenerate initiator consensus sequence has been defined: (5’) Y-Y-A+1 -N-T/A-Y-Y-Y (3’)
where A+1 is the base at which transcription starts, Y is a pyrimidine (C or T), N is any of the four bases, and T/A is T or A at position +3. u Transcription of many protein-coding genes has been shown to begin at any one of multiple possible sites, often 20200 base pairs; such genes give rise to m. RNA with multiple alternative 5’ ends.
u Most genes of this type contains a CG-rich stretch of 20 -50 nucleotides within =100 base pairs upstream of the start-site region. u The presence of a CG-rich region, or Cp. G island, just upstream from a start site is a distinctly nonrandom distribution.
u The SV 40 enhancer stimulates transcription from all mammalian promoters when it is inserted in either directions anywhere. u Enhancers are located 50 or more kilobases from the promoter they control. u Different eukaryotic cellular enhancers
have shown that they can occur upstream from a promoter, downstream from a promoter within an intron, or even downstream from the final exon of a gene.
u The start site at which transcription initiates encodes the first 5’ nucleotide of the first exon of an m. RNA, the nucleotide that is capped. u For those encoding abundantly expressed proteins, a TATA box located approximately 25 -35 base
pairs upstream from the start site directs RNA polymerase II to begin transcription at the proper nucleotide. u Promoter-proximal elements (=10 base pairs), are located within the first =200 base pairs upstream of the start site. Enhancers are about
50 -200 base pairs long and are composed of multiple elements of =10 base pairs. u Enhancers may be located up to 50 kilobases or more upstream or downstream from the start site or within an intron. u More than one enhancer regions.
u The S. cerevisiae genome contains regulatory elements called upstream activating sequences (UASs), which function similarly to enhancers and promoter-proximal elements in higher eukaryotes. Most yeast genes contain only one UAS, which generally lies within a few
hundred base pairs of the start site. In addition, S. cerevisiae genes contain a TATA box=90 base pairs upstream from the transcription start site.
u The preinitiation complex is an association of RNA polymerase II and several protein initiation factors that assemble together at the start site and begin to unwind the DNA in preparation for transcription of the gene.
u RNA polymerase II requires the addition of several initiation factors. u These initiation factors, which position polymerase molecules at transcription start sites and help to melt the DNA strands so that the template strand can enter the active site of the enzyme, are called general transcription factors.
u The general transcription factors that assist Pol II in initiation of transcription from most TATA-box promoters. u These proteins are called TFIIA and TFIIB…. and most are multimeric proteins; the largest is TFIID, which consists of a TATA-box binding
protein (TBP) and 13 TBP-associated factors (TAFs). u TBP is the first protein to bind to a TATA-box promoter. All eukaryotic TBPs have very similar C-terminal domains of 180 residues. u The N-terminal domain of TBP, which varies greatly in sequence
and length among different eukaryotes, functions in the Pol IIcatalyzed transcription of genes encoding sn. RNAs. u TBP interacts with the minor groove in DNA, bending the helix considerably. The DNA-binding surface of TBP→ TATA promoter.
u TBP has bound to the TATA box, TFIIB can bind. u TFIIB is slightly smaller than TBP. u The C-terminal domain of TFIIB makes contact with both TBP and DNA on either side of the TATA box, while its N-terminal domain
extends toward the transcription start site. u Following TFIIB binding, a preformed complex of tetrameric TFIIF and Pol II binds, positioning the polymerase over the start site. u At most promoters, two more
general transcription factors must bind before the DNA duplex can be separated to expose the template strand. u First to bind is tetrameric TFIIE, creating a docking site for TFIIH, another multimeric factor
containing ten subunits. u