Exam 4

Where is TATA box located, and what nucleotides is it majorly composed of?

-30, T&A

What are basal transcription machinery for prokaryotes?

-35 & -10 consensus sequences, sigma factor, promoter

Briefly describe steps of Splicing.

1. 2' OH of Branch-point Adenosine attacks at 5' splice site, right at boundary between intron and exon. It creates 3'OH at end of exon. During this step, intron forms lariat structure (looped form of RNA) 2. 3'OH at the end of first exon attacks 3' splice site, where it joins 2 exons. It also releases the intron. lariat gets degraded.

What are post-transcription modifications?

1. 5' G cap (7 methyl cap) 2. splicing to remove introns (and maybe some exons too)* a gene does not always have introns. 3. poly-A tail is added at 3' end.

What are the steps of Transcription in eukaryote?

1. A region of the DNA gets transcribed, and produces pre-mRNA. 2. pre-mRNA gets processed. 3. mature RNA is transported out to cytoplasm to be translated.

List three similarities between prokaryotic and eukaryotic transcription.

1. Both has RNA polymerase(s) which moves 5' -> 3' direction. 2. both has promoter sequence (which signals where to start and gives direction). 3. Both has some kind of termination mechanism.

How many RNA polymerases does Eukaryotes have?

3: RNA polymerase I, II, III

Which direction is the message (mRNA) read?

5' -> 3', just like transcription and translation.

What is RNA polymerase II CTD composed of? What are the characteristics of it?

52 copies of NSNNSNN (S stands for Serine- 7 amino acids). It is perfect repeat of 52 copies, and it is also unstructured.

What is a start codon, and which Amino Acid does it code for?

AUG, and Methionine

What is 7' methyl G cap capped by?

Cap Binding protein (CBC)

(T/F) Consensus sequence is conserved for prokaryotes and eukaryotes.

False. Consensus sequence is different for prokaryotes and eukaryotes.

(T/F) One prokaryotic organisms uses only one transcription termination pathway.

False. For example, E. coli uses both ways, however, there are different genes that use different termination pathway.

What is Monocistronic mRNA, and what type of organism has it?

Monocistronic mRNA is when there are only one gene per one pre-mRNA, and eukaryotes have monocistronic mRNA.

Which direction is the AA sequence read?

N-terminus to C-terminus, or Amino-end to carboxyl-end (same thing)

Why is splicing ATP-intensive?

Splicing uses ATP to bring conformational changes

What are basal transcription machinery for eukaryotes?

TATA & BRE element, promoter

Describe what TFIIH is composed, and what it does.

TFIIH has kinase, helicase and ATPase, which helps TFIIH to open the helix.

What does other sequences that are not promoter sequences bind to, and what kind of function does it serve?

They are bound to proteins that serve their functions, and different sequences allows the ability of the gene to be regulated deferentially depending of cell types, such as control on frequency of transcription.

What is a possible explanation why prokaryotes don't have introns?

existence of intron means the pre-mRNA has to go through splicing, but splicing would have been disturbance to their rapid division.

Fill in the blank. CBC, EJC, and PAB recruit a(n) ________________________________.

export receptor. You need ALL THREE to recruit export receptor.

Why was discovery of universal genetic code important at the time (and now)?

it got translation to work in extract, and allowed the scientists to use simplified RNA template.

Where are promoter sequences located in prokaryotes??

-35 and -10 bp (+1 is the start of transcription)

Where is BRE sequence located, and what nucleotides is it majorly composed of?

-35, C&G

Describe steps of polyadenylation with proteins involved.

1. Endonuclease is recruited, and it cleaves off the RNA at polyadenylation site. The last 5' end of RNA unused (intron )is degraded by 5'->3' exonuclease. 2. Poly A polymerase (PAP) comes in, where it adds 200-250 As at the end of the message. 3. Poly A tail is bound by poly A binding protein (PAB) which helps it to protect RNA.

What is the importance of CBC?

1. Regulates nuclear export. If there is no appropriate G-cap present, mRNA cannot exit nucleus. 2. Prevents degradation y 5'->3' exonuclease. 5'->3' exonuclease usually get rid of useless RNA. However, G-cap makes mRNA "looks like" 3' end, which ends up protecting mRNA. 3. Promotes translation. Without any modifications, it cannot exit nucleus.

List 6 Transcriptional factors in Eukaryote in order of them being used.

1. TFIID 2. TFIIB & TFIIA 3. TFIIF 4. TFIIE 5. TFIIH

What is the other name of promoter sequences?

Consensus sequence.

What does difference between two promoter sequences tell the RNA polymerase?

Direction of polymerase (depends on location of -35 and -10 sequences: which strand they are located)

Fill in the blank. After splicing & leave of spliceosome, ________________________________ replaces at break between exons.

Exon Junction Complex (EJC)

Describe what TFIIB (and TFIIA) does.

It has one component which recognizes BRE on DNA. it helps position the complex correctly on the promoter. In other words, it helps the RNA polymerase to get orientation.

What is the benefit of having a polycistronic mRNA?

It helps the cell to easily regulate the production of proteins, because the proteins produced from one polycistronic mRNA should most like be regulated the same way.

Describe what is TFIID composed of, and what it does.

It is composed of TAT binding protein (TBP) which is responsible for recognizing TATA box, and 11 other TBP-associated factors (TAFs). Together bind to DNA, which causes 90 degree bend in DNA. This 90 degree bend puts strain on DNA, which helps RNA polymerase to open the helicase.

What is "Snurps"?

It is small nuclear ribonuclear protein, which is composed of snRNA (small nuclear) and proteins.

What does EJC does?

It marks successful splicing, where cell uses it to check the quality of splicing.

What does export receptor provide to overall transcription?

It provides quality control. For example, if Snurps are still there, mRNA gets degraded (since it means that splicing was not done completely)

Describe what TFIIF does.

It recruits RNA polymerase II.

What does transcriptional factors form?

Pre-initiation complex

(T/F) Gene include regulatory sequences.

True

(T/F) poly-pyrimidine tract is located at 3' splice site.

True.

(T/F) Most exons have similar size.

True. (100-500 nt)

(T/F) 3'-splice site is located at 5' end of exon.

True. 5' end of exon is adjacent to 3' end of intron, where 3'-splice site is located.

(T/F) Downstream of the gene is located after the end of transcription.

True. Downstream of the gene is located at 3' end of the gene.

(T/F) For one region of DNA, transcription usually occurs in one direction.

True. However, Transcription can occur both direction along the chromosome.

(T/F) Regulatory region is located relatively far away from region that produces RNA.

True. It can also be located near region that produces RNA.

Fill in the blank. ________________________________ recognizes 5' splice site and ________________________________ recognizes 3' splice site and branch point sequence through specific base pair interactions between Snurps & RNA sequence on splice sites.

U1, U2

Fill in the blank. ________________________________ comes together after Snurps recognized splice site which brings adenosine to 5' splice site.

U4, U6 and U5

List three STOP codons.

UGA, UAA, UAG

When does addition of 7' methyl G cap happen?

When 25-30 nt has been transcribed.

What are the things that RNA polymerase has to know before it starts its job?

Where to start transcription, which direction it has to go, where to stop...

Fill in the blank. Universal genetic code exists prior to ________________________________ of all organisms.

last common ancestor

Fill in the blank. Export receptor interact with ________________________________ to facilitate export to cytoplasm.

nuclear pore complex

What kinds of sequences are at the ends of a gene, and why is it important for a gene to have it?

regulatory sequences are on both ends of the gene, and regulatory sequences are important to gene expression.

Fill in the blanks. Splicing is catalyzed by several ________________________________ complexes comprising the ________________________________.

ribonucleoprotein, spliceosome.

What is TATA box recognized by?

specific initiation factors, composed of more than 100 proteins

Describe prokaryotic transcription cycle.

1. Sigma factor slides along DNA (it has weak interaction with DNA) to search for promoter sequence. Once it finds the sequence, it binds to the major groove of the sequence. 2. Sigma factor forms the complex at promoter, where it opens the double helix of DNA. At this point, the RNA polymerase is ready to start transcription, as RNA polymerase does not need primer to start. 3. RNA polymerase starts transcription; it generates short piece of RNA (which often disassociate), but starts again. This is because sigma factor is bound to promoter too hard; therefore even RNA polymerase wants to transcribe, it cannot physically go forward. 4. RNA polymerase does scrunching motion. RNA polymerase pulls DNA into itself to break free from promoter. At this point, sigma factor disassociates, and it is free to go other places to recruit RNA polymerase. RNA polymerase is finally going through elongation. 5. At the end of the gene, RNA polymerase terminates.

List three functions of Poly A tail.

1. it Affects message stability 2. Aids in nuclear export 3. Aids translation. If you remember, this is exactly same list of functions that G-cap serve.

Describe steps of 7' methyl G cap addition.

1. phosphatase: it removes most terminal phosphate (goes from 3 phosphate -> 2 phosphate) 2. Guanylyl transferase: creates 5'-5' linkage which flips guanine to attack. 3. Methyl transferase: it adds methyl group, specific to C7 of guanine at the end. 4. another enzyme: add methyl group to 2' OH.

What is Basal transcriptional machinery?

Base minimum for transcription to happen in Eukaryotes: This is NOT sufficient for most of the time, because Eukaryotic transcription happens in terms of chromatin, where prokaryotes don't have chromatin

Which splice-site is branch-point sequence close to, and what does it have that is important to splicing?

Branch point is close to 3'-splice site(18-40 nucleotides away), and it has invariant Adenosine.

Fill in the blank. RNA processing steps are coordinated by RNA polymerase II ________________________________.

C-terminal tail (CTD) RNA polymerase C-terminal Tail is a catalytic subunit of RNA polymerase.

(T/F) 5'-splice site is located at 5' end of exon.

False. 5'-splice site is located at 5' end of intron.

(T/F) length of poly A tail does not matter in terms of stability; the existence of poly A tail helps the mRNA to not be degraded.

False. Longer Poly A tail means the mRNA has longer half life. This can be effective in terms of gene regulation.

(T/F) most of the splice-site sequences are located in exon.

False. Most of it is located in intron, and 3 nucleotides in total is located in intron (don't need to memorize this)

(T/F) There are two ways where pre-mRNA gets processed: addition of poly A tail and G-cap.

False. Pre-mRNA also gets processed through splicing.

(T/F) RNA stay paired with DNA until it has to come apart for transcription bubble to move along.

False. RNA does not stay paired with DNA.

(T/F) RNA polymerase for prokaryote is low processive, therefore it requires additional protein to help it not fall off.

False. RNA polymerase for prokaryote is processive, therefore it can make long mRNA without falling off.

(T/F) Sigma factor is used for both prokaryotic and eukaryotic transcriptions.

False. Sigma Factor is only used for prokaryotic transcription. Eukaryotic transcription uses a lot more complex initiation pathway.

(T/F) Template strand and sense strand are the same strand.

False. Template strand gets transcribed, where sense strand does not get transcribed.

(T/F) Most introns have similar size.

False. Their sizes vary by a lot. (many kilobases)

(T/F) Promoter sequence is usually enough for both prokaryotic and eukaryotic transcription.

False; prokaryotic transcription usually only requires promoter, but eukaryotic transcription requires additional regulatory sequences and proteins.

(T/F) Universal Genetic Code is same in ALL organisms.

False; there are some variations in mitochondria and protosomal organisms. Other than these organisms, Universal Genetic Code is same.

What kind(s) of RNA does Gene codes for?

Functional RNA and messenger RNA.

Describe how phosphorylation of RNA polymerase II CTD helps mRNA post-transcription modifications.

G-cap: Serine 5 in number of repeat are phosphorylated by TFIIH at initiation. This recruits capping enzyme. This happens when 25nt-30nt of transcription is done. Splicing: Serine 2 is phosphorylated by phosphorylation of serine 5. BOTH phosphorylated Serine 2 & 5 recruits splicing proteins. Poly A-tail: de-phosphorylation of serine 5 (partially) recruits 3' processing factors. All three processes are coordinated processes. CDC is also 10 times longer than diameter of main portion of RNA polymerase.

Describe intrinsic mechanism of transcription termination, and which organism uses this?

Intrinsic mechanism only needs RNA to terminate. At the site of termination, there is inverted repeat, where sequence of RNA that has self complementary. This forms a hairpin structure, which stops transcription after self-complimentary sequence is transcribed. U residues follow after hairpin structure, which have relatively weak base pairing. At this point, RNA polymerase falls off. Prokaryotes use this mechanism for termination. (1 way)

What is the difference between original and current definition of Gene?

Original definition of Gene was "a unit of inheritance", where current definition of Gene is more specific to RNA. When original definition of Gene was developed, sciences were only able to visualize gene with phenotype.

What is difference between Poly A polymerase (PAP) and RNA polymerase II?

Poly A polymerase does not require a template strand, where only thing it can utilize is As. Therefore, As at the end of mRNA are NOT encoded by DNA. Also, Poly A polymerase is DNA polymerase. RNA polymerase II requires template strand, like other polymerases.

Fill in the blank. ________________________________ of mRNA processing and transcription termination are linked in Eukaryote.

Polyadenylation

What is Polycistronic mRNA, and what type of organism has it?

Polycistronic mRNA is where one gene produces one mRNA sequence, but one mRNA can produce multiple different proteins. This is possible because there are distinct regions on an mRNA that produce different proteins. These proteins often serve functions in a same pathway. Prokaryotes have polycistronic mRNA.

What is abortive initiation, and what goes through this and when? What happens after?

Pre-initiation complex for prokaryotic and eukaryotic transcription goes through abortive initiation, where few nucleotides are added, try to be attached, fail, removed, and another comes in. Once it is successful in euk, TFIIH stimulates promoter release, where transcription factors leave to start transcription. Elongation factors are recruited, it joins with RNA polymerase. Elongation factors keep mRNA moving, therefore they proceed in transcription. This is still basal level.

Why are there no proofreading in transcription?

RNA is not getting passed from one cell to another, therefore when the cell makes a mistake during transcription, other correct copies can be made through different round of transcription. In other words, it is not that important for cells to fix error during Transcription. Therefore, Transcription is a lot more error-prone (1 in 10^4 bp)

Describe three types of RNA polymerase.

RNA polymerase I: transcribes rRNA production, where it constitute part of ribosome. RNA polymerase II: transcribes mRNA, which is produced from protein encoding genes. It also transcribes miRNA (micro RNA: involved in gene regulation) and non-coding RNA (telomerase RNA, snRNA (involved in splicing)). RNA polymerase III: transcribes tRNA (transfer RNA), one of rRNA (5s RNA), and other small functional RNA

Fill in the blank. ________________________________ of spliceosome provide recognition of sequences through base pairing, and ________________________________ of spliceosome catalyze splicing.

RNA, RNA&protein together

What is the current definition of Gene?

Region of DNA that encodes instructions to make RNA

Describe Rho dependent mechanism of transcription termination, and which organism uses this?

Rho is a helicase protein, where it has 5' to 3' movement on RNA. it recognizes certain sequences, so it binds to RNA at the site. It starts moving along the RNA, following RNA polymerase. Rho usually have similar speed with RNA polymerase, therefore it does not hinder the transcription. However, when hairpin structure forms on RNA, it stops RNA polymerase. Rho eventually catches up, and it unwinds RNA and DNA complex, which eventually releases RNA polymerase and mRNA. Prokaryotes use this mechanism for termination. (1 way)

What are consensus sequences bound by in prokaryote before transcription, and which specific groove does it bind to?

Sigma Factor, and it binds to major groove for specificity.

What is RNA polymerase in prokaryotes bound to on DNA, what benefit does it serve to transcription, and how?

Sigma factor, and it opens up the DNA by going through conformational change

What is one reason why splicing requires so much machinery and ATP?

Splicing is high fidelity process, since if even one base pair is off during splicing, the entire mRNA goes wrong. Therefore, many machinery and ATP is required to make sure that the processes don't go wrong.

What is the possible explanation why eukaryotes have introns?

Splicing makes it easy for cells to have different combinations of exons to produce different proteins.

What are the similarities between DNA polymerase and RNA polymerase?

They both have 5' -> 3' polarity (3' OH on existing RNA strand attacks 5' phosphate on incoming nucleotide)

(T/F) Multiple RNA polymerase can work at the same time over same region.

True

(T/F) introns are generally larger than exons.

True

(T/F) A lot of gene is transcribed but don't end up in final mRNA.

True, since introns which are not in final mRNA are much longer than exons.

(T/F) Eukaryotes have more steps than prokaryotes in relation to production of protein.

True.

(T/F) RNA-like strand and coding strand are the same strand.

True.

(T/F) if you put E.coli promoter in eukaryotic cell, it wouldn't work.

True.

(T/F) There are NO helicase involved in transcription.

True. Instead, RNA polymerase creates transcription bubble where it opens DNA strand for 10-12 base pairs. except rho dependent transcription termination: uses rho as protein)

(T/F) Upstream of the gene is located at 5' end of the gene.

True. Upstream of the gene is the region before start of transcription.

What kinds of information does promoter sequences tell the RNA polymerase?

Which direction to go and Where to start

Fill in the blank. When there are multiple codons that code for 1 AA, 3rd position is ________________________________.

Wobble Position