At some promoters RNA polymerase cannot initiate transcription without assistance from an ancillary protein
- these proteins called positive regulators because necessary to switch on the transcription unit
- example CAP (catabolite activator protein) prevents use of other sugars by E. coli in medium where glucose is also present (operons for enzymes involved in utilisation of lactose, arabinose, galactose etc are switched off in presence of glucose)
- CAP is necessary to initiate transcription at dependent promoters
and is only active in presence of cAMP (cyclic AMP)
Fig 18.6 (Concepts)
- when there is high level of glucose in cell, cAMP levels is reduced and when glucose conc’n is low, cAMP level is high
- when cAMP levels are low, CAP is unable to bind to control region operon and this prevents RNA pol from binding
- CAP functions to turn off alternative pathways when they become unnecessary because the cell has an adequate supply of glucose
- binding sequence for CAP is
T G T A G A
or similar and a CAP dimer binds most strongly to sites that contain two inverted versions of the pentamer seq
- CAP also interacts directly with the "-subunit of RNA polymerase
Fig 18.9 (Concepts)
e.g. in the trp operon of E. coli
a mechanism that controls the ability of RNA polymerase to read through an ATTENUATOR, which is an intrinsic terminator located at the beginning of a transcription unit
- 2E structure (folding) of RNA transcript may (under certain conditions) result in formation of the hairpin needed for intrinsic termination
- if the termination hairpin forms, transcription is terminated before structural genes are transcribed
- if hairpin is prevented from forming, RNA pol transcribes structural genes
- termination at the attenuator responds to the level of tryptophan. In the presence of adequate amounts of trp, termination is efficient and in the absence of trp RNA pol can continue into the structural genes
- repression at the trp operon responds in the same way
- when trp is present operon is repressed and RNA polymerases that escape from the promoter then terminate at the attenuator. when trp is removed RNA pol has access to the promoter and does not terminate prematurely
how does termination at the attenuator respond to the level of trp?
- note alternative base-pairing of regions 1, 2, 3 and 4: region 2 is complementary to 1 & 3, region 3 is complementary to 2 & 4
- pairing of regions 3 and 4 generates the hairpin terminator
- in the leader region the initiation codon (AUG) is followed by an open reading frame (coding region) that contains two successive Trp codons (UGGUGG)
- when trp is present, charged tRNA trp is available and ribosomes are able to synthesize the leader peptide. the ribosomes continue along region 1 and extend over region 2
- this prevents region 2 from base pairing . region 3 can then base pair with region 4 generating the terminator hairpin
- RNA polymerase then terminates at the attenuator
- when trp is absent, charged tRNAtrp is unavailable and ribosomes stall at the Trp codons in region 1. region 1 then cannot pair with region 2.
- regions 2 and 3 can base pair before region 4 is transcribed and region 4 remains single-stranded. In the absence of the terminator hairpin (region 3 base paired with region 4) RNA pol continues past the attenuator
- thus ribosome stalling influences termination at the attenuator
- transcription and translation must be coupled and the leader must be translated in order for the hairpin to form
- the attenuator provides a mechanism for sense the supply of Trp-tRNA and therefore responds directly to the need of the cell for tryptophan in protein synthesis
- attenuation has a ~10x effect on protein synthesis as it only allows ~10% RNA pols to proceed in presence of trp and allows almost all RNA pols to proceed when trp is absent
- when repression is releases in absence of trp effect is ~70x increase in transcription
- these two mechanisms together allow ~700-fold range of regulation of the trp operon
- attenuation also found in at least 6 operons coding for biosynthesis of aa including his and leu
All these activities together can coordinate extremely complex and delicate
developmental regulatory systems. Each gene or transcription unit may be
simple but the whole picture may be complicated.
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
This template created by the Web Diner.