11 - Transcription of Genes Flashcards
antisense strand
Strand of DNA that is complementary to the coding seq and is used as a template for mRNA. noncoding strand/template strand
the sense strand is the strand that is identical to the mRNA (except the DNA/RNA thing), but is not used as a template.
RNA, like DNA, is synthetizes 5’->3’ direction.
Housekeeping genes
constitutive genes. expressed under most conditions. required for the fundamental operations of the cell.
terminology
cistron = segment of DNA/RNA that encodes a single polypeptide chain.
ORF = any seq of bases that could, in theory, encode a protein.
monocistronic mRNA = mRNA carrying the information of a single cistron, which is a coding seq for only a single protein.
operon = a cluster of prok genes that are transcribed together to give a single mRNA (polycistronic mRNA)
polycistronic mRNA = mRNA caarryinf the coding seq (cistrons) for several proteins
structural gene = seq of DNA/RNA that codes for a protein or for a nontranslated RNA molecule.
transcription start terminology
promoter = the seq to which RNA polymerase binds
5’ UTR = region of an mRNA between the 5’ end and the translation start site
3’ UTR = seq at the 3’ end of mENA, downstream of the final stop codon, which is not translated into a protein
Transcription beginning recognition
promoter = where RNA polymerase binds
3’ UTR = end of mRNA, not translated. downstream of final stop codon.
5’ UTR = between 5’ end of mRNA and translation start site
Bacterial transcription
- 10 (-35) region = region of bacterial promoter 10 (35) bases back from the start of transcription that is rec by RNA polymerase
- 10 region is AT rich - easier to unwind the double helix here than if CG rich
Pribnow box = -10 region (named after its discoverer - not really used anymore)
bacterial RNA polymerase consists of two major components - the core enzyme (5 subunits, alpha, alpha, beta, beta’ and omega) and the sigma subunit. Together, they make up the holoenzyme.
core enzyme = responsible for RNA synthesis.
Sigma subunit = responsible for recognizing promoter. Rec -10 and -35 regions of the coding (nontemplate) strand.
consensus seq for -10: TATAAT
consensus seq -35: TTGACA
(in E. coli)
The strenght of the promoter depends partly on how closely it matches the consensus seq.
Manifacturing the message (bacteria)
1) sigma subunit binds to promoter
2) RNA polymerase core enzyme opens up the DNA helix locally to form a transcription bubble.
3) a single strand of RNA is sythetized using one DNA strand as template. the sigma subunit is no longer needed and often (not always) detaches.
The first transcribed base of mRNA is usually an A, flanked by two pyrimidins (often CAT). Sometimes it can be G, but almost never a pyrimidine.
Core enzyme of bacterial RNA polymerase
5 subunits: 2x alpha beta beta' omega
beta + beta’ = catalytic site
alpha required partly for assembly and partly for recognizing promoters
omega binds beta’, stabilizes it and aids in its assembly into the core enzyme complex.
RNA polymerase has a deep groove through the middle (can accomodate ca 16 bp of DNA in bacteria (25 i euks)). A thinner groove, roughly at right angles to the first, may hold the new RNA.
Prok RNA polymerase is inhibited by the antibiotic rifampicin, which binds to the channel for DNA and RNA within the beta-subunit of the enzyme, physically blocking elongation. Defence = single nt mutation (lower affinity for rifampicin)
RNA polymerase knows where to stop
terminator = stop signal
in template strand, consists of two inverted repeats separated by half a dozen bases and followed by an A-run. The A-run results in a string of Us in the mRNA. the inverted repeats means that a stem-and-loop structure can form, causing RNA polymerase to pause, the string of Us and As are bound weakly, and the RNA and DNA fall apart during the pause. (if there is no string of Us then it will keep going, it happens in long mRNAs).
Two classes of terminators exist: Rho-dependent and -independent (intrinsic). Rho is a protein that separates the RNA polymerase and DNA (in rho-dependent). Rho-independent dont need no protein to do dis.
Rho protein
can sepaarate DNA and RNA polymerase (in rho-dependent terminators).
specialized helicase
uses energy from ATP to unwind
consists of a hexamer (six identical subunits that recognize and binds to seq 50-90 bases upstream of the terminator in the mRNA).
RNA seq for Rho binding = rut seq. poorly defined, but high in C and low in G. 6 clustered rut seq is needed (one per subunit).
Upon binding, it changes its conformation to a doughnut, causing allosteric changes in the catalytic subunits of RNA polymerase, causing termination. Rho then unwinds the RNA/DNA helix and separates the two strands.
How does a cell know which genes to turn on
genes that are only needed in certain conditions have poor match with consensus seq in -10 and -35. It thus needs an activator protein to induce transcription. these activator proteins are different for different genes, and can stimulate one or more genes transcription. genes activated by the same protein will be expressed together under similar conditions, even if they are at different locations in the DNA.
What activates the activator?
E. coli example:
maltose can be used to satisfy the E. colis needs. An activator protein, MalT, detects maltose and binds it. This causes the protein to change shape, exposing its DNA binding site (the original nonbound form cannot bind DNA). The active form (MalT + maltose) binds to a specific seq iof DNA found only in the promoter regions of genes needed for growth on maltose. The presence of MalT helps RNA polymerase bind to the promoter and transcribe its genes.
The small molecule (here maltose) that induces gene expression, is called an inducer.
negative regulation of gene transctiption in bacteria
positive regulation: activator protein binds gene when the gene is to be turned on
negative regulation: repressor protein binds the DNA and ensures that the genes is turned off. if the repressor is removed, the gene can be transcribed. repressors bind to operator seq.
Like inducers, repressors alternate between DNA-binding and nonbinding forms.
multiple mathods: steric hinderance at promoter, bind within the structural gene (RNA can bind but not start transcription). In the LacI case, both the repressor and the RNA polymerase bind DNA at the same time, causing RNA polymerase to bind more tightly and thus not move.
LacI protein
repressor.
if no lactose, LacI binds to operator (overlaps part of the promoter and the front part of the coding region for the lactose-genes).
if lactose: LacI changes shape, cannot bind, released, lactose genes are transcribed.
Many regulator proteins bind small molecules and change shape
allosteric protein = protein that changes shape when it binds a small molecule
signal molecule = small molecule that exerts a regulatory effect by binding to a regulatory system
if the repressor affects some nutrient-stuff, the nutrient is often the signal molecule.
signal molecule makes the regulator protein change shape.
regulator proteins have an even nnumber of subunits
Transcription in Euks is more complex
multiple RNA polymerases (usually 3, each transcribing a different category of genes, as well as separate RNA polymerases in mitochondria and chloroplasts)
RNA polymerase I:
- transcribes the genes for the two large rRNA molecules + 5.8 rRNA.
RNA polymerase III
- transcribeds the genes for tRNA, 5S rRNA, and several other small RNA molecules.
RNA polymerase II
- transcribes most euk genes that encode proteins, and as a result is subject to the most complex regulation
RNA polymerase I and III are constitutively active in mist genes, as tRNA and rRNA is needed all the time.
the RNA polymerases have subunits.
Rpol II has 10, 3 of which are shared with Rpol I and III. the largest subunit of Rpol II is related to the beta’ of bacterial, and posesses the carboxy-terminal domain (CTD) tail. The assorted TFII complexes each consist of several polypeptide chains.
TFs
general and specific
general = needed for the transcription of all genes transcribed by a particular RNA polymerase.
nomenclature: TFI, TFII, TFIII + letter (depending on which Rpol.
specific TFs are needed for transcription of particular genes under specific circumstances (proteins sych as the sigma subunit of bacterial Rpol may also be regarded as TFs, however this terminology usually only applies for euk).
Transctiption of rRNA and tRNA in euks
Genes for the large rRNAs are present in multiple copies (7 in E. coli and several hundred in higher euks). In proks they are dispersed (spredt), whereas they are in clusters of tandem repeats in euks.
in humans: clusters on 5 chromosomes. the genes for 18S, 5.8S and 28 S rRNA form a single mRNA (by Rpol I) to give a single large RNA (45 S pre-rRNA). The long transcript is then cleaved to release the 3 separate rRNA molecules.
synthesis of rRNA by Rpol I is localized to a zone in the nucleus calles the nuleolus. here, the rRNA precursor is transcribed and processed. The rRNAs then bind proteins, giving ribonucleoprotein (RNP) particles.
the promoter for Rpol I is CG-rich (uncommon due to H-bonds). There are 2 CG rich regions, the core promoter and the upstream control element, both of which are rec by UBF1 (upstream binding factor 1, single polypeptide binding a homodimer). After binding, selectivity factor SL1 binds (several polypeptides). Once UBF1 and SL1 are in place, TIF1A helps Rpol I bind.
UFB1 has both specific and general roles.
1) binds throughout the region of DNA that encodes a cluster of tandem rRNA genes and keeps this region in an open configuration.
2) at the individual promoters UBF1 binds in the upstream region and, in cooperation with SL1, bends DNA around in a loop that probably makes contact with other factors as well as Rpol I.
Rpol III makes 5S rRNA and tRNA, in addition to some small nuclear RNAs (snRNAs).
Promoters for 5S and tRNA are unique and internal (!!!) to the genes. Trc requires the binding of either TFIIIA or TFIIIC to a region over 50 bp downstream from the start site. binding of these allow TFIIIB to bind and start trc.
Transcription protein-encoding genes in euks
enhancers = reg seq that binds TFs but is outside (and often far away from) the promoter.
Initiator box = seq at the start of trc of an euk gene
primary transcript = RNA molecule produced by trc before it has been processed in any way
TATA box = binding site for a T that guides Rpol II to the promoter in euks
upstream element = DNA seq upstream of the TATA box in euk promoters that is rec by specific proteins
TBP = TATA binding protein, binds TATA box, needed for binding of Rpol I and III. Binds in minor groove (unusual). TATA is surrounded by CG rich regions.
TBP forms TIID (needed to rec promoters specific for Rpol II. Binding of TFIID to TATA via TBP is the first step of transcription initiation, though several other TFs are needed as well. After TFIIA and B bind, Rpol II can bind, but cannot move from the promoter (known as RNA pausing). the moving from the promoter requires 3 more TFs.
Upstream elements increase the efficiency of RNA pol II binding
5-10 bp long
50 to 200 bp upstream
TFII = general TFs (always required).
most specific TFs don’t make contact with Rpol directly, but rather via TFIIs (usually TFIID). They help the assembly of the transcription apparatus and therefore increases the frequency of trc.
Common upstream elements include CG box (GGGCGG), CAAT box, AP1 element.
During mRNA elongation, RNA pol is also subject to neg reg, like by NELF (negative elongation factor)
Enhancers control trc from a distance
Especially during development
usually cluster of rec sites, can therefore bind several proteins.
Usually a long distance away from the genes they control (thousands of bp). Upstream or downstream.
make contact with trc apparatus
when an enhancer switches a gene on, it makes a loop in the DNA so that the enhancer is closer to the gene.
transcription in archaea (357-359, tror ikke det er spesielt viktig men vi får se)
in terms of molecular biology, archaea are closer to euks than bacteria. However, the regulation of trc resembles that of bacteria.
