Differences between euk and eubacterial genes
- (promoters, leader sequences, RNA polymerases, introns)
- promoter sequences different
- one multiunit RNA polymerase in eubac, 3 multiunit polymerases in
euks
- some euk genes have internal promoters
- presence of introns in euk coding sequences
- any combination of signals that control transcription
- many euk genes have unique promoter signals which specify (i) transcription start site, (ii) frequency of initiation of transcription and (iii) maximum rate of transcription
- these respond to environmental stimuli, cell signals (e.g. hormonal responses) & specific stages in development pathways of the organism
Some promoter signals are more generally found e.g. TATA (-25) and CAAT (-75) boxes which have the consensus seqs: TATAAAA (euk pol II) and GGCCAATCT
mutation of TATA box sequence leads to variation of transcription start point, i.e. TATA function s as positioning component of promoter
In proks: T80 A95 T45 A60 A50 T96 (-10, Pribnow)
T82 T84 G78 A65 C54 A45 (-35)
RNA Polymerases
3 in euks
POL I transcribes rRNA (except 5s rRNA)
POL II transcribes mRNA & small nuclear RNA (snRNA)
POL III transcribes tRNA, 5s rRNA & small cytoplasmic RNA (scRNA)
pol III genes have INTERNAL CONTROL REGIONS (ICRs) rather than 5' (upstream) promoters - promoters are within the genes themselves.
begins with 5' purine, usu A
5' UTR (UnTranslated Region) variable between diff genes, e.g.
- 35 bp in human immunoglobulin lambda light chain gene
- 670 bp in rat HMG CoA reductase gene
during mRNA formation (20-30 b), the 5'ATP ( or GTP, first transcribed base) is enzymatically modified by addition of N7 methylated guanosine (nucleoside) to give a m7G-pppA cap to the message
note 5'-ppp-5'
cap becomes attached to cap binding protein during migration from nucleus.
mRNA + protein ribonucleoprotein (RNP)
protein component involved with alignment at SSU and is aided by small cytoplasmic RNPs (SCYRPs, scRNPs).
coding sequences called exons
discovered by e.m. analysis of heteroduplex DNA
variable numbers & sizes of introns
- none in human " interferon genes, >50 in rat "-2 procollagen gene
usu 75 bp to 2 kb but 17 kb intron in rat thyroglobulin gene, approx 80 kb in some Drosophila genes
relatively large primary mRNA transcript (pre-mRNA) translated to smaller protein, e.g. human factor VIII clotting protein has 9 kb of exons in 186 kb sequence.
in yeast there are 233 introns on 16 chr & 6000 genes (most yeast genes do not have introns)
Intron splicing in nuclear genes
5'-G T . . . . A G-3' rule
consensus in 5' - 3' antisense strand of DNA
DNA
exon intron exon
A G G T A A G T. . . . . . . C A C T G A C . . . . . . . 6Py N C A G N
left, 5' branch site right, 5'
consensus at 5' end
5' A64 G75 G100 T100 A62 A68 G84 T63
consensus at branch site (18-40 bp upstream of 3' end)
in yeast T A C T A A C
Py80 N Py80 Py87 Pu75 A100 Py95 invariant A100
consensus at 3' end
6Py74-87 N C65 A100 G100 (12 Py in yeast)
RNA
5' A G G U A A G G U . . . . . . .C A C U G A C . . . . . .6Py N C A G N
Overall, cleavage occurs at the 5' exon/intron boundary (left, or donor site) generating a 5' guanosine
-this G loops back and interacts with the penultimate A in the branch site
- forms a loop held together by a 5' - 2' linkage
- cleavage occurs at right boundary (acceptor site)
- exons are joined together and intron is released as free lariat
cis-acting sequences that increase utilisation of (some) eukaryotic
promoters, increase efficiency of transcription, generally <20 nt
can function in either orientation and in any location (upstream or
downstream) relative to the promoter, not gene specific
similar to UAS in prokaryotes except UAS always upstream
contains several closely arranged sequence elements that bind transcription
factors
may be responsible for tissue-specific transcription e.g. Ig genes
that carry internal enhancers which are active only in the B lymphocytes
in which the Ig genes are expressed
enhancers appear to function by localization of the protein bound at
the enhancer to increase its chance of contacting proteins that bind the
promoter
this may necessitate DNA folding or looping as enhancers are often
hundreds of bp from the promoter
genetic switching may be regulated by competition for an enhancer
e.g. the switch from foetal (-haemoglobin to adult $-globin polypeptide
chains. Enhancer changes preference during development
Fig
cis-acting sequences which depress or down regulate the efficiency adjacent genes are known as silencers
in euk genes of variable length
3'UTRs of around 50-200 nucleotides
some longer, e.g. 520 nt in rat "-2 crystallin gene
also some genes have no 3'UTR
- a gene may have >1 trailer, e.g. mouse dihydrofolate reductase gene
has 4 transcript classes with different 3'UTRs varying from 80-930 nt
- these trailers may be concerned with gene expression (regulation
of gene expression includes post-transcriptional mRNA stability
pre-mRNA mRNA
- 3'UTRs may contain sequences that bind proteins and regulate decay/stability
of mRNA
- e.g. in Xenopus oocyte maturation, translation is enhanced by protein
interaction with specific signals in some 3'UTRs of mRNAs.
In many (most) euk genes the 3' flanking region is transcribed and
translation stops at specific sequences. Then the 3' end of the primary
transcript is cleaved by endonuclease function of CFI and CFII (cleavage
factors).
cleavage occurs downstream of a signal in the UTR, this is known as
the pol A signal and has the consensus
A A U A A A
in the primary transcripts of higher euks (but not in yeast)
- this signal is recognised and bound by a specificity component (CPSF - cleavage-polyadenylation specificity factor) that directs the cleavage.
- a tract of polyadenosine phosphate residues is enzymatically added by poly(A) polymerase (polyadenylase) - about 11to 30 nt downstream of the CPSF recognition site and this forms the poly A tail.
Most transcripts have polyA tails added except some of the histone mRNAs, e.g. H3 mRNA where formation of the 3' end depends on secondary structure - RNA terminates on a stem-loop structure by complementary base-pairing and on a sequence that base pairs with the U7 snRNA.
poly A tails usu ~ 200 nt, but poly A tails in yeast are ~ 50 nt.
Fig
Regulation of Gene Expression in Eukaryotes
a number of ways (Fig 19.1, Concepts of Genetics)
(1) Transcription factors and enhancers can control the rate of DNA transcription
- the majority of regulatory events occur at the initiation of transcription
- the gene may be regulated by a sequence at promoter or enhancer that is recognised by a specific protein (transcription factor) needed for RNA pol to initiate
Some TFs control many genes, e.g. in the heat shock response - an increase in temp turns off transcription of some genes and turns on transcription of heat shock genes
- these genes share a common consensus sequence called HSE (heat shock
element) and may contain multiple copies of it. HSE conserved from Drosophila
to mammals. Consensus is:
CNNGAANNTCCNNG
and is bound by HSTF (heat shock transcription factor)
- a gene may have several different elements located in an enhancer or promoter that can activate the gene
(e.g. human metallothionein gene)
Proteins that interact with DNA and regulate transcription may have
certain motifs that are responsible for binding to DNA
e.g. (a) ZINC FINGER originally recognised in TFIIIA (for pol III), a DNA binding domain now known in other DNA binding proteins
Sequence of ‘finger’ is:
Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His
i.e. Cys2/His2
Fig 22.13 (Principles of Genetics)
Cys2/Cys2 also present in TFIIB and steroid receptors (TFs that become
active upon binding steroids)
Fig 19.16 (Concepts of Genetics)
(b) HELIX-LOOP-HELIX (HLH) PROTEINS
- motif is a sequence of 40-50 aa that contains two "-helices (DNA binding) separated by a linker region (loop) of varying length
- usually contain a basic region adjacent to the HLH motif
- HLH proteins can bind DNA as homodimer or heterodimer
Fig 22.13 (Principles of Genetics)
(c) LEUCINE ZIPPERS (see handout)
- stretch of aa rich in leucine residues that provide a dimerisation motif
bZIP motif is amphipathic helix where leucines of one protein interact
with those of another while basic regions of proteins bind DNA
Fig 22.13 (Principles of Genetics)
Codon usage bias
Organisms exhibit a property known as codon preference
- of the two or more degenerate codon possibilities that exist for a particular aa, an organism/species will preferentially use one more frequently than it uses the others
- there will be more of the tRNA in the cell and a preferred set of charged tRNAs
- mRNAs that have a number of the rarely used codon will be translated inefficiently because of the tRNA limitation
(3) Alternate splicing pathways
e.g. in the human fibronectin gene, differential splicing may generate up to 10 different polypeptides
e.g. troponin T mRNA in mouse has one form in smooth muscle and another
in other tissues by attaching exon 1 to either exon 2 or exon 3.
(4) mRNA stability
can be regulated by 3'UTRs (as previous)
(5) Post-translational processing
can produce different proteins from a single-chain precursor polypeptide
- e.g. several different pituitary hormones can be produced in different tissues of the pituitary from the same starting polypeptide by different cleavage
(6) Different 3'UTRs and polyadenylation sites
Can affect splicing at the 3' end of the transcript leading to polypeptides with different CARBOXYL terminus sequences and different properties
- e.g. heavy chains of IgM can be membrane bound or secreted depending
on the carboxyl terminus
(7) Different N-termini of polypeptides
- can act as signal sequences
- different mRNA initiation sites can produce different leaders which can also determine where the finished polypeptide will go.
- the signal seq would be in the leader
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