protein synthesis: transcription Flashcards
define transcription
the process by which single-stranded RNA sequences are formed from double stranded DNA templates
what are the 3 stages of transcription?
initiation, elongation, termination
what enzyme synthesises the RNA strand in prokaryotes?
RNA polymerase holoenzyme
what enzyme synthesises the RNA strand in eukaryotes?
RNA polymeraase I, II and III
primarily II (as synthesises mRNA)
broadly, how is transcription regulated in prok vs euk?
prok: physical blocking of RNA Pol binding sites
euk: enhancers
what are the other functions of RNA polymerases, besides the formation of the RNA strand?
search the double stranded DNA to find promoter region to bind and initiate synthesis
unwind a short stretch of helical DNA to produce 2 single strands
select complementary rNTP to the one on the template strand + catalyse formation of phosphodiester bonds
backtrack to correct sequence errors
which strand does the polymerase bind to
template (anti-sense)
how does RNA synthesis differ between organisms?
chemically identical in prok and euk
differ in posttranscriptional processing + regulation and type of polymerase used
what is the core promoter region for prokaryotes?
-35 (TTGACA)
-10 (TATAAT) (Pribnow box)
[ the consensus sequences ]
what is present in addition to the core promoter region in genes that are highly expressed?
another interaction site for pol alpha subunit between 40 and 60 nucleotides upstream of the TSS
what is needed for the initiation of transcription in prok?
just the RNA polymerase holoenzyme
what is common to both eukaryotic and prokaryotic RNA polymerases?
have 2 similar, large subunits
contain central Mg2+ or Mn2+ near a large cleft on 1 side
what is the composition of the core enzyme RNA pol in prok?
2 alpha, beta’, beta, omega
α subunits are structural, assembling the holoenzyme and associated regulatory factors
β subunit contains the polymerase activity that catalyzes the synthesis of RNA
β’ subunit nonspecifically binds to DNA.
ω subunit is involved in assembly of the holoenzyme + maintaining structural integrity of the RNA pol
sigma is associated
what is the role of the sigma subunit in RNA pol ?
used to recognize the promoter decreasing the affinity of RNA Pol to DNA in general, but increasing the affinity of RNA Pol for specific DNA promoter sequences
also allows RNA pol to scan for promoters as holoenzyme has lower affinity for DNA
why are there multiple forms of sigma subunit?
they recognise different promoter sequences
when does the sigma subunit dissociate?
after initiation
where is RNA Polymerase I found and what does it produce?
nucleolus
rRNA : 5.8S, 18S and 28S
why is RNA polymerase II important?
responsible for synthesising mRNA and producing all precursor RNA that will be translated into ppcs + proteins
also synthesises snRNAs and miRNAs (crucial for post-transcriptional and post0-translational modifications)
what does RNA polymerase II use as its promoter complexes?
TATA box
initiator elements
downstream promoter elements (DPEs)
what is the function of RNA polymerase III?
makes tRNA and a little snRNA and 5S rRNA
where is transcription initiated in eukaryotes?
TATA box
or CAAT or GC box in housekeeping genes
what is required for the initiation of transcription in eukaryotes?
RNA polymerase II, general transcription factors to assemble promoter machinery
describe the TATA box binding protein
30 kDa component of TFIID
saddle shaped protein with 2 domains
concave surface binds TATA box, inducing conform changes + widening of minor groove, thus beta strands can contact helix
why does TBBP ensure formation of an asymmetric promoter complex?
ensures transcription proceeds unidirectionally
what is the order of recruitment of the promoter complex?
TFIID recruits TFIIA and TFIIB and TFIIF with RNA pol II
then TFIIE and TFIIH to form basal transcriptional machinery
(see gene expression cards)
what does TFIIH do?
opens DNA helix and phosphorylates carboxyl-terminal domain, allowing enzyme to dissociate from promoter and beginning elongation
where are promoter sequences found in eukaryotes?
downstream core promoter element (DPE) around 28-32 nucleotides downstream
does not matter since recognised by proteins other than RNA polymerase so don’t have to be specific size
in which direction is RNA synthesised?
5’ to 3’
where does the energy for unwinding of DNA come from?
interactions of DNA while wrapped around RNAP, stabilising single strands and helping pull the template strand through the enzyme ..?
what causes RNA polymerases to unbind from DNA?
termination signal
(or unreliable binding in the first 10 pairs)
how many base pairs long is the transcription bubble?
17
what is contained in the transcription bubble?
RNA polymerase
nascent RNA
8 base pair long RNA-DNA hybrid helix
how is the RNA positioned in the transcription bubble?
so that the 3’ OH group on its most recently added nucleotide can attack the α-phosphorous atom of the next incoming ribonucleoside triphosphate
how might RNA polymerase be able to proofread its own transcription?
might backtrack, go opposite way to elongation
3’ to 5’ to detect non-watson-crick base pairs
to break NWC pair is less energetically costly as bases are not complementary and don’t form the right number of H bonds
RNAP puts last-but-one phosphodiester bond level with Mg2+ of active site: hydrolysis cleaves bonds and releases dinucleotide containing incorrect terminal nucleotide
why is a mistranscription not such a big problem?
many copies of each transcript made and effects are not passed on through generations
how can transcription be terminated in prokaryotes? where do the functioning signals lie?
rho-dependent and rho-independent
in the nascent RNA strand
how was rho-dependent termination discovered?
during in vitro experiments : some RNA molecules synthesized by RNA Polymerase in isolation were longer than when they are transcribed in vivo
proposed that Rho is necessary for proper termination when hairpin loop structures are not present
method of action of rho elucidated when rho was added to an incubation mixture immediately after, a few seconds after and 2 minutes after RNA synthesis had been initiated.
yielded 10S, 13S and 17S RNA molecules, and if no rho was added, a 23S molecule.
Evidently, rho detects additional termination sites not detected by RNA Polymerase alone, of which this molecule contains 3, as well as 1 rho-independent site, yielding the 23S product.
how does rho-dependent termination work?
helicase (rho) terminates it
rho = hexameric helicase, hydrolyses ATP in presence of single-stranded DNA but not double
activated by sequences of RNA that contain many cytosines and few guanines
rho binds to RNA and pulls it through its centre, breaks the RNA-DNA hybrid helix once it catches the transcription bubble
how does rho-independent termination work?
physical modified RNA structure terminates it
inverted palindromic repeats that get transcribed into nascent RNA (CCGG….GGCC followed by AT rich region)
creates a self-complementary RNA transcript that forms H bonds between C and G to form hairpin loop
followed by at least 4 Us: forming rU-dA bonds (weakest type possible)
RNAP pauses here, nascent RNA dissociates from DNA and then enzyme, closing transcription bubble
how does termination occur in eukaryotes?
RNAP II makes AAUAAA (and GU??): polyadenylation signal
activates + summons cleavage enzymes, cleaving RNA away from enzyme and DNA
then up to 250 As are added to the 3’ end
what post-transcriptional modifications does RNA undergo in eukaryotes?
addition of polyA tail to 3’ end
addition of 7-methyl guanosine cap to 5’ end
splicing to remove non-coding introns
what is alternative splicing?
when different combinations introns are removed to create an overall different mRNA to be translated
why is alternative splicing useful?
allows 1 pre-mRNA to code for multiple proteins that are variations on a theme
one gene can code for many similar proteins such as cell receptors or antibodies
why is the ability of eukaryotic cells to regulate precisely the time at which each gene is transcribed crucial?
enabled multicellular organisms to evolve with distinct tissues
what 3 factors influence gene expression in eukaryotes?
nuclear membrane : transc + transl occur in diff compartments: spatial + temporal separation
complex transcriptional regulation : enhancers + variety of promoter elements compared to prok
RNA processing : extensive processing of pre-mRNA
how are tRNA transcripts processed?
5’ leader cleaved by RNase P
3’ trailer is removed and CCA added by CCA-adding enzyme
intron is removed by endonuclease
how are rRNA transcripts processed?
pre-rRNA modified on both ribose and base components directly by snoRNPs
assembled with ribosomal protiens
3 rRNAs cleaved from each other
in the nucleolus
how is the 5’ end of pre- mRNA modified?
phosphoryl group released by hydrolysis
remaining diphosphate attacks alpha-phosphorous of GTP to form 5’-5’ triphosphate linkage : the cap
N-7 nitrogen of terminal guanine methylated to form cap 0
how do caps work to protect mRNA?
protect 5’ ends from phosphatases and nucleases
enhance translation of mRNA
how is the 3’ end modified?
AAUAAA recognised by endonuclease
250 As added using ATP as donor
what is the role of the polyA tail?
enhances stability and translation efficiency of mRNA
determines in part the half life of an mRNA by its rate of degradation
what is small nuclear ribonucleoprotein (SNRP)? what does it do?
snRNA and proteins together
cleaves out introns in pre-mRNA and stitches together exons to make mature mRNA
How do SNRPs work?
bind to specific nucleotide site on intron, near 3’ of first exon and 5’ of second exon (GU at beginning, 10 pyrimidines then AG at end)
SNRP cleaves at GU leaving exon 1 with 3’ OH unbound
2’-OH of adenylate residue in branch site attacks 5’ terminal phosphate of the intron = 2’-5’ phosphodiester bond between them
> generates a branch
3’-OH of exon 1 attacks phosphodiester bond between intron and exon 2
exons 1 and 2 become joined
2 transesterification reactions (number of phosphodiester bonds stays the same: reaction can proceed without energy source ATP or GTP)
what are some diseases caused by improper splicing?
spinal muscular atrophy
hypotonia/hypoflexia
beta thalassemia
what is the attacking group in the splicing reaction?
2’-OH of adenylate residue in the branch site
what is the composition of the spliceosome?
large ribonucleoprotein
snRNAs bind to proteins to form small nuclear riboprotein complex (snRNP)
many combine to form spliceosome
what is the purpose of the spliceosome?
removes introns from transcribed pre-mRNA
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