Lecture 9 - Eukaryotic Transcription

Question 1

how many RNA polymerases are there in prokaryotes?

Answer 1

in prokaryotes there is a singular RNA polymerase

Question 2

how many RNA polymerases are there in eukaryotes and what are their functions?

Answer 2

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

Question 3

how do eukaryotic RNA polymerases work?

Answer 3

work in the same way in synthesising RNA at an enzymatic reaction between nucleotides, however they do require assistance in binding to the promoters

Question 4

what is the TATA box?

Answer 4

the TATA box is a sequence of about 25 upstream of the transcription start site that has the sequence TATAAAA

Question 5

what is the function of the TATAAA sequence upstream of the transcription start site?

Answer 5

it is the recognition sequence for the TATA box-binding protein (TBP) which is within the transcription factor II D complex (TFIID)

Question 6

TBP and its structure relationship to function:

Answer 6

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

Question 7

what does the binding of TBP and TFIID to the TATA box allow for?

Answer 7

this allows for the recruitment of additional proteins to bind, and these are called general transcription factors

Question 8

where do general transcription factors bind?

Answer 8

general transcription factors bind to additional upstream control elements which are present in the 200 base pairs upstream of the transcription start site

Question 9

transcription factor structure:

Answer 9

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

Question 10

what do general transcription factors help with?

Answer 10

the placement of the RNA polymerase II to the promoter and for the transcriptional start site

Question 11

order of general transcription factor binding:

Answer 11

unknown, thought to differ between genes

Question 12

enhancers and repressors at long distances away from the promoter can affect…

Answer 12

… the stability of the RNA polymerase II/general transcription factor complex at the promoter

Question 13

in order to have an effect on transcription, these cis-acting regulator proteins act through…

Answer 13

… mediator complexes which bind the RNA polymerase II/general transcription factors

Question 14

how can regulator binding affect action at the promoter?

Answer 14

can increase rate of transcription up to 1000-fold

Question 15

what is required for the promoter to be exposed for transcription?

Answer 15

chromatin remodelling

Question 16

how does DNA being wrapped around a nucleosome affect transcription?

Answer 16

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

Question 17

what is transcription factor II H?

Answer 17

actually a complex of proteins, with each individual part playing important roles in the initiation of transcription

Question 18

both of these steps are mediated by transcription factor II H:

Answer 18

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

Question 19

Both steps of melting and ATP hydrolysis are mediated by proteins within TFIIH:

Answer 19

– 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

Question 20

what does the DNA & melting and ATP hydrolysis create?

Answer 20

the transcription bubble where the DNA strands are separated to allow for transcription to occur

Question 21

RNA Polymerase II Escapes the Promoter:

Answer 21

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

Question 22

for mRNA destined for translation, three modifications occur in order to keep RNA stable and ready for translation:

Answer 22

– Capping
– Splicing
– Polyadenylation

Question 23

in order to protect the RNA transcript from degradation and to enable nuclear export and translation the:

Answer 23

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

Question 24

when does capping occur?

Answer 24

capping occurs after 25 nucleotides have been added to the RNA transcript

Question 25

introns:

Answer 25

non-coding sequences that exist in genes as important regulatory sequences

Question 26

introns:

Answer 26

non-coding sequences that exist in genes as important regulatory sequences

need to be removed before translation so only exons remain

Question 27

splicing can occur on the pre-mRNA while transcription is occurring as long as:

Answer 27

the 5’ end has been capped

Question 28

Introns are Numerous and Long:

Answer 28

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

Question 29

why does transcription take far longer in eukaryotes in comparison to prokaryotes?

Answer 29

as a result of introns, mRNA transcription takes significantly longer in eukaryotes than in bacteria

Question 30

Splicing Adds Diversity to Gene Expression (+ why its useful):

Answer 30

• 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.

Question 31

what do introns always start and end with?

Answer 31

Introns always start with a GU on the 5’ end and always end with an AG on the 3’ end

Question 32

at each end of the introns in the primary transcript are:

Answer 32

splice site indicating the boundaries of the intron

Question 33

intron branch site:

Answer 33

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

Question 34

Splicing Forms Branches Structures for Removal:

Answer 34

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

Question 35

where does splicing occur?

Answer 35

splicing all occurs in the spliceosome – a complex protein and RNA structure that is responsible for undertaking the cutting of the transcripts

Question 36

what does the spliceosome consist of?

Answer 36

structure has small nuclear RNAs (snRNA) and over 150 different proteins

Question 37

because snRNAs are involved in mediating the bringing together of the splice and branch sites, what is it similar to?

Answer 37

an enzyme

Question 38

the gene SMN1 is duplicated with SMN2 which is the same sequence apart for:

Answer 38

a different nucleotide in an important splicing site that removes exon 7

Question 39

SMN2:

Answer 39

produced a non-functional version of the protein

Question 40

spinal muscular atrophy (SMA):

Answer 40

spinal muscular atrophy is a group of diseases that is caused by a loss of SMN1 that decreases motor neurone survival

Question 41

what can cause SMA (muscular spinal atrophy)?

Answer 41

mutations that affect transcription, translation and/or splicing can cause spinal muscular atrophy

Question 42

why are RNA transcripts are capped on the 5’ end?

Answer 42

to maintain stability of the transcript

Question 43

how are the introns from genes spliced out from the primary RNA transcript?

Answer 43

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

Question 44

why is splicing important?

Answer 44

splicing is important in providing genetic diversity in expression of genes in eukaryotes

Question 45

when will RNA polymerase II terminate transcription?

Answer 45

RNA polymerase II will reach a termination sequence where it will stop transcribing and synthesising RNA

Question 46

what will RNA polymerase II do before it dissociated from the DNA and RNA?

Answer 46

before it disassociates from the DNA and RNA will transcribe the polyadenylation signal which allows for enzymes involved in these processes to bind

Question 47

what do polyadenylation signals do?

Answer 47

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

Question 48

what are the functions of the poly(A) tail?

Answer 48

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

Question 49

when is RNA transported for translation?

Answer 49

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

Question 50

when is transcrition fully complete?

Answer 50

with the cleavage and polyadenylation of the RNA transcript

Question 51

what are the poly(A) tails with the 5’ caps important features for?

Answer 51

they are important features for: stability, transport and for translation

Question 52

what are general transcription factors critical for?

Answer 52

general transcription factors are critical for initiation of transcription in eukaryote

Question 53

what does the TATA-binding protein within the TFIID act like?

Answer 53

sigma factors in bacteria

Question 54

what structural elements can affect the bonding of RNA polymerase II to the promoter?

Answer 54

structural elements such as chromatin and distant regulatory sequences

Question 55

TFIIH:

Answer 55

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