Transcription in Eukaryotes
Transcription in Eukaryotes
Transcription in Eukaryotes
A. Carried out by three different RNA polymerases, each of which contains more than10
subunits (i.e., these are very complex enzymes). All three require several assisting proteins,
collectively called transcription factors.
RNAP I → all ribosomal RNAs (5.8S, 18S, 28S) except for 5S found in the
nucleolus
RNAP II → all nuclear genes encoding proteins (mRNAs) found in the nucleus
RNAP III → all tRNAs, the 5S rRNA, and all snRNAs found in the nucleus
1. For RNAP II (protein-coding genes), initiation requires several transcription factors that
assist binding to promoter sites. Promoters sites recognized by RNAP II (and associated
protein factors) are several conserved elements that are located upstream from the
transcription start point (the +1 base). The consensus sequences of the conserved
elements are…
c) GGGCGG [GC box] & often present but occur in different positions and in
ATTTGCAT [octamer box] different copy numbers
2. The TATA homology is found in all eukaryotic promoters known to date. The
remaining “consensus” sites are found but not necessarily in the same promoter. All
“consensus” sites affect binding efficiency of RNA polymerase/transcription factors.
3. RNAP I and RNAP III utilize some of the same transcription factors as RNAP II but the
promoters are quite different. RNAP III utilizes internal promoters (i.e., within the
transcriptional units).
1. Elongation of the RNA chain is similar to that in prokaryotes except that a 7-methyl
guanosine (7-MG) cap is added to the 5’ end when the growing RNA chain is fairly
short (20-30 bases in length).
(a) The 7-MG cap is “attached” by an unusual 5’-5’ triphosphate linkage and serves to
protect the growing RNA from degradation by nucleases. This “capping” is part of
RNA processing in eukaryotes.
(b) Cleavage occurs 10-30 bases downstream from the conserved sequence AAUAAA.
(c) After cleavage, an enzyme [poly(A) polymerase] adds about 200 adenine (A) bases
to the 3’ends. This is called polyadenylation or the addition of poly-A tails.
(i) The function of poly-A tails is to increase stability of the transcript and to
assist in transport of the mRNA from the nucleus to the cytoplasm. This is
another part of RNA processing is eukaryotes.
2. Termination of transcription via RNAPI and RNAP III is via response to discrete
termination signals.
1. These are sequences that exist upstream or downstream of the transcribe sequence, and
that enhance or depress rates of transcription.
2. Most enhancers and silencers are not well characterized, can exist close to or distant
(>1,000 bp) from the gene(s) they affect.
A. General facts:
1. Most eukaryotic genes are “split” (have intervening sequences), including protein-
coding genes and tRNA & rRNA genes. Exceptions include histones and a few others.
2. There are a few introns in prokaryotes. Most are found in viruses and an archebacteria.
4. In general, the amount of intron sequence per “gene far exceeds the amount of exon
sequence. Some examples are given in the text; others are listed below.
5. Some introns can be quite large. One intron in the Ubx gene of Drosophila, for
example, is roughly 70,000 bp in length.
6. The only features shared in common by all introns in protein-coding genes are splice
sites.
B. Splicing: there are three distinct types of intron excision, one for tRNA, one rRNA, and
one for mRNA.
(i) Involves five small snRNA molecules (U1, U2, U4, U5, & U6) that range
in size from 100 to 215 nucleotides
(iii) snRNAs do not exist as free RNA molecules but rather are complexed
with several proteins into snRNPs (small nuclear ribonucleoproteins)
(b) The initial step is cleavage at the 5’ intron splice site (↓GU-intron), followed by
intramolecular phosphodiester linkage between the 5’ carbon of the G residue at
the cleavage site and the 2’ carbon of a conserved A residue near the 3’ end of the
intron, forming a lariat structure.
(c) The next step is cleavage of the 3’ splice site, followed by joining of the two
exons via a 5’ to 3’ phosphodiester linkage.
(d) The spliced, processed mRNA is then transported to the cytoplasm for translation.
C. Why introns?
(b) Sequence differences between introns (even within the same species) are large,
indicating that there is little constraint on intron sequence per se.
2. The most interesting speculation about possible function of introns is exon shuffling.
(a) Most, proteins have several domains. These domains include substrate
recognition, cofactor recognition, catalytic regions, allosteric functions, et cetera.
Examples previously discussed include the DNA polymerases (e.g., DNAP-I, the
Kornberg enzyme).
(b) Many proteins (e.g., enzymes) share one or more domains, and via “exon
shuffling” new “genes” coding for similar but different proteins could evolve
through recombination of different domains (exons). The evolutionary advantage
would be to make “new” genes into single transcriptional units.