Lecture 9 - Eukaryotic Transcription Flashcards
how many RNA polymerases are there in prokaryotes?
in prokaryotes there is a singular RNA polymerase
how many RNA polymerases are there in eukaryotes and what are their functions?
in eukaryotes there are three RNA polymerases which are responsible for transcribing different RNA classes:
–RNA polymerase I and III transcribe rRNA and tRNA genes
–RNA polymerase II transcribes mRNA and some other non-coding RNA genes
how do eukaryotic RNA polymerases work?
work in the same way in synthesising RNA at an enzymatic reaction between nucleotides, however they do require assistance in binding to the promoters
what is the TATA box?
the TATA box is a sequence of about 25 upstream of the transcription start site that has the sequence TATAAAA
what is the function of the TATAAA sequence upstream of the transcription start site?
it is the recognition sequence for the TATA box-binding protein (TBP) which is within the transcription factor II D complex (TFIID)
TBP and its structure relationship to function:
important as this DNA-binding protein has two similar structures that allow it to bind to the DNA and causes the DNA to bend
this bending allows for the recruitment of additional transcription factors to help recruit RNA polymerase II to bind
what does the binding of TBP and TFIID to the TATA box allow for?
this allows for the recruitment of additional proteins to bind, and these are called general transcription factors
where do general transcription factors bind?
general transcription factors bind to additional upstream control elements which are present in the 200 base pairs upstream of the transcription start site
transcription factor structure:
they have two domains that bind to DNA regulatory regions and the RNA polymerase through other proteins
they bind to the major groove of the DNA, hence how promoter provides strand specificity
what do general transcription factors help with?
the placement of the RNA polymerase II to the promoter and for the transcriptional start site
order of general transcription factor binding:
unknown, thought to differ between genes
enhancers and repressors at long distances away from the promoter can affect…
… the stability of the RNA polymerase II/general transcription factor complex at the promoter
in order to have an effect on transcription, these cis-acting regulator proteins act through…
… mediator complexes which bind the RNA polymerase II/general transcription factors
how can regulator binding affect action at the promoter?
can increase rate of transcription up to 1000-fold
what is required for the promoter to be exposed for transcription?
chromatin remodelling
how does DNA being wrapped around a nucleosome affect transcription?
if the DNA is wrapped around a nucleosome, then none of the general transcription factors are unable to bind to the TATA box and initiate transcription
what is transcription factor II H?
actually a complex of proteins, with each individual part playing important roles in the initiation of transcription
both of these steps are mediated by transcription factor II H:
molecule helps to separate the strands for RNA Polymerase II
ATP hydrolysis is required for the separation of the DNA strands and the initiation of transcription
Both steps of melting and ATP hydrolysis are mediated by proteins within TFIIH:
– one of the proteins that makes up TFIIH called a helicase pries the double helix apart and melts the DNA at the transcription start site
– this is done by using the energy obtained by hydrolysing ATP generated by an ATPase that is part of TFIIH
what does the DNA & melting and ATP hydrolysis create?
the transcription bubble where the DNA strands are separated to allow for transcription to occur
RNA Polymerase II Escapes the Promoter:
RNA Polymerase II is bound to all these DNA-binding proteins and held in place, to escape this two kinases that are part of the TFIIH phosphorylates the amino acids at the C-terminal of the protein
this C-terminal sequence is specific to eukaryotes and is not present in bacteria
this phosphorylation allows RNA Polymerase II to disengage from the general transcription factors and begin to elongate the RNA transcript
for mRNA destined for translation, three modifications occur in order to keep RNA stable and ready for translation:
– Capping
– Splicing
– Polyadenylation
in order to protect the RNA transcript from degradation and to enable nuclear export and translation the:
5’ end of the RNA is capped - the cap involves the removal of the terminal phosphate group and the addition of a guanine to form a 5’ to 5’ triphosphate bridge
this guanine is then methylated at the N7 position, and sometimes additional methylation can occur
when does capping occur?
capping occurs after 25 nucleotides have been added to the RNA transcript
introns:
non-coding sequences that exist in genes as important regulatory sequences
introns:
non-coding sequences that exist in genes as important regulatory sequences
need to be removed before translation so only exons remain
splicing can occur on the pre-mRNA while transcription is occurring as long as:
the 5’ end has been capped
Introns are Numerous and Long:
Introns are often longer than the exons themselves and sometimes longer than all exons together
–In humans, exons averaged 150 nucleotides, while introns average 1,500 nucleotides
why does transcription take far longer in eukaryotes in comparison to prokaryotes?
as a result of introns, mRNA transcription takes significantly longer in eukaryotes than in bacteria
Splicing Adds Diversity to Gene Expression (+ why its useful):
• splicing allows for different versions of RNA transcripts and consequently proteins to be produced from the same gene
• this can be useful is different forms of the protein, called isoforms, are needed in different times and/or cells.
what do introns always start and end with?
Introns always start with a GU on the 5’ end and always end with an AG on the 3’ end
at each end of the introns in the primary transcript are:
splice site indicating the boundaries of the intron
intron branch site:
found within the intron which is focused around a single A, which is important for excision and is normally close to the 3’ end and the 5’ end
Splicing Forms Branches Structures for Removal:
the 2’ OH group on the A that is part of the branch site bends the transcript and performs a nucleophilic attack on the first G of the 5’ splice site, which causes it to be cut
this frees the 5’ end of the intron then becomes covalently linked to the 2’ OH of the A on the branch point -forming what is called the lariat intermediate
the OH group of the 5’ exon then does a nucleophilic attack itself on the last G in the 3’ splice site
– this forms the intron lariat which is removed from the mRNA
– it also joins up the two exons together to be part of the mature RNA transcript
where does splicing occur?
splicing all occurs in the spliceosome – a complex protein and RNA structure that is responsible for undertaking the cutting of the transcripts
what does the spliceosome consist of?
structure has small nuclear RNAs (snRNA) and over 150 different proteins
because snRNAs are involved in mediating the bringing together of the splice and branch sites, what is it similar to?
an enzyme
the gene SMN1 is duplicated with SMN2 which is the same sequence apart for:
a different nucleotide in an important splicing site that removes exon 7
SMN2:
produced a non-functional version of the protein
spinal muscular atrophy (SMA):
spinal muscular atrophy is a group of diseases that is caused by a loss of SMN1 that decreases motor neurone survival
what can cause SMA (muscular spinal atrophy)?
mutations that affect transcription, translation and/or splicing can cause spinal muscular atrophy
why are RNA transcripts are capped on the 5’ end?
to maintain stability of the transcript
how are the introns from genes spliced out from the primary RNA transcript?
through the spliceosome, the introns from the genes are spliced out from the primary RNA transcript though a series of nucleophilic attacks of OH groups on set sequences present in splices sites at the boundaries of introns
why is splicing important?
splicing is important in providing genetic diversity in expression of genes in eukaryotes
when will RNA polymerase II terminate transcription?
RNA polymerase II will reach a termination sequence where it will stop transcribing and synthesising RNA
what will RNA polymerase II do before it dissociated from the DNA and RNA?
before it disassociates from the DNA and RNA will transcribe the polyadenylation signal which allows for enzymes involved in these processes to bind
what do polyadenylation signals do?
these signals will cleave the RNA to expose the 3’ polyadenylation site
Poly-A polymerase (PAP) will then bind to the 3’ end of the transcript and add around 200 adenines without a template
The addition of the poly(A) tail helps to provide stability to the mRNA and to promote translation
what are the functions of the poly(A) tail?
in adding around 200 adenines to the template it helps not only in providing stability to the mRNA to promote translation but they are also important for translation as:
(1) the 5’ cap binding protein recognises the 5’ cap
(2) multiple poly(A) binding proteins bind along the entirety of the poly(A) tail
when is RNA transported for translation?
once capped, spliced and polyadenylated, the RNA is exported from the nucleus with all the proteins attached
with the proteins attached, this is a signal to the nuclear pore complex to move the RNA transcript from inside the nucleus to the cytoplasm where it can be translated
when is transcrition fully complete?
with the cleavage and polyadenylation of the RNA transcript
what are the poly(A) tails with the 5’ caps important features for?
they are important features for: stability, transport and for translation
what are general transcription factors critical for?
general transcription factors are critical for initiation of transcription in eukaryote
what does the TATA-binding protein within the TFIID act like?
sigma factors in bacteria
what structural elements can affect the bonding of RNA polymerase II to the promoter?
structural elements such as chromatin and distant regulatory sequences
TFIIH:
protein complex that is required to melt the DNA strands apart to allow RNA polymerase to attach to the DNA to initiate transcription, as well as phosphorylating RNA polymerase II to enable it to escape the promoter
