Transcription & RNA Processing 1 (Non-mRNA)

Question 1

What are 4 ways in which eukaryotic transcription differs from prokaryotic transcription?

Answer 1

1. In eukaryotes, DNA is packaged with proteins in a complex called chromatin. Chromatin that is wound tightly together makes it difficult for RNA polymerases to access the DNA.
2. There are 3 RNA polymerases in eukaryotes.
3. Transcription occurs in the nucleus, and the RNA template must be moved to the cytosol for translation to occur.
4. Transcription factors account for roughly 10% of eukaryotic genes, implying the importance of transcriptional regulation.

Question 2

What are housekeeping genes?

Answer 2

Housekeeping genes are constitutive genes that are required for basic cellular structure and function.

Question 3

What is meant by constitutive expression?

Answer 3

Most cell types express a common set of proteins so they all have constitutive (the same) expression of basic cell structure and central metabolism to the same degree in all tissues.

Question 4

What are tissue specific genes?

Answer 4

Tissue specific genes code for cell-specific proteins that determine the phenotype of the cell. This process is highly regulated.

Question 5

What is an example of cell-specific protein?

Answer 5

Globin is only located in RBCs.

Question 6

What does RNA polymerase 1 transcribe?

Answer 6

RNA polymerase 1 transcribes the precursors of large ribosomal RNAs:
5.8S rRNA, 18S rRNA, 28S rRNA

Question 7

What does RNA polymerase 2 transcribe?

Answer 7

RNA polymerase 2 transcribes mRNA (messenger that will become protein), miRNA (micro), most snRNA (small nuclear that is involved in splicing).

Question 8

What does RNA polymerase 3 transcribe?

Answer 8

RNA polymerase 3 transcribes small genes like tRNA (transfer) 5S rRNA (ribosomal), U6 snRNA (small nuclear).

Question 9

What are chloroplast and mitochondrial polymerases transcribe?
To what are they similar?

Answer 9

Chloroplast and mitochondrial polymerases transcribe chloroplast genes and mitochondrial genes.
They are similar to bacterial enzymes.

Question 10

What is the structure of the RNA polymerases?

Answer 10

The RNA polymerases have 12 to 17 sub-units. They all have 9 core sub-units, 5 of which closely match the prokaryotic core sub-units.

Question 11

What do RNA polymerases require?

Answer 11

Eukaryotic RNA polymerases require General Transcription Factors (GTFs) that are distinct from the polymerase and its core subunits to initiate transcription.

Question 12

What is special about RNA polymerase 2?

Answer 12

Only RNA polymerase 2 transcripts are polyadenylated.

Question 13

What are the elements of eukaryotic RNA transcription promoters?

Answer 13

1. TATAA box: spans -25 to -30 bases, similar to the Pribnow element in prokaryotes, located in 10 to 20 % of RNA polymerase 2 promoters, critical for GTF binding to start initiation
2. Inr (initiator) elements: span transcriptional start sites
3. BRE (B Recognition element): located at -35 bases, recognizes TFIIB
4. Downstream elements: DPE, DCE, MTE
   Combinations of these elements bind general transcription factors.

Question 14

How many GTFs are required to for initiation of eukaryotic RNA transcription?

Answer 14

At least five general transcription factors are required for initiation of transcription in vitro.

Question 15

What is the Initiation process of eukaryotic RNA transcription?

Answer 15

1. TFIID (a complex of 15 proteins) binds to TBP:
   Certain chains recognize specific promoter elements, such as TBP (TATA binding protein) and other peptides within TFIID called TAFs (TBP associated factors).
2. TFIIB binds to the BRE promoter element and TBP.
3. RNA polymerase 2 binds to TFIIF.
4. The TFIID-TBP complex binds the RNApol2-TFIIF complex.
5. TFIIE and TFIIH bind the complex, completing the pre-initiation complex. (TFIIH has two subunits that are helicases, XPB and XPD.)
6. Another TFIIH subunit phosphorylates the 5th serine residue in a repeated sequence on the C Terminal Domain (CTD) of RNA polymerase 2.
7. RNA polymerase 2 dissociates from the complex and transcription begins!

Question 16

What two TFIIH subunits are helicases?

Answer 16

XPB and XPD are helicase subunits of TFIIH.

Question 17

What is Mediator?

Answer 17

Mediator is a protein complex that has more than 20 subunits and is involved in transcription.

Question 18

What are the 2 function of Mediator?

Answer 18

1. Mediator interacts with both GTFs and RNA polymerase 2 for transcription to take place.
2. Mediator interacts with gene specific transcription factors and “mediates” gene specific control of transcription initiation.

Question 19

When does Mediator leave the Initiation Complex?

What happens next?

Answer 19

1. Mediator leaves the initiation complex when initiation has occurred and the CTD of RNA polymerase 2 is phosphorylated.
2. The CTD then binds other proteins that facilitate elongation.

Question 20

What else happens when TBP binds the TATA box?

Answer 20

TBP puts a kink in the RNA strand.

Question 21

What is the other model of RNA transcription initiation?

How does this model differ from the step-wise model?

Answer 21

The recruitment of preassembled complex model suggests that the RNA polymerase 2 preforms a complex with all of the GTFs. This complex is then recruited to specific promoters by activation factors so that it can bind in together with the DNA to initiate transcription.

Question 22

How is compaction a form of RNA transcription initiation regulation?

Answer 22

The compaction process blocks binding of RNA polymerase 2 to the promoter and confers regulation to the promoter and initiation complex.

Question 23

How is histone acetylation a form of RNA transcription initiation regulation?

Answer 23

Histone acetylation removes the charge on lysine residues, thus dissociating nucleosomes from DNA. This results in less dense chromatin that is more accessible to RNA polymerase 2 and allows for RNA transcription initiation.

Question 24

What happens after the full length of the RNA molecule is transcribed?
Where does this occur?

Answer 24

1. After the full length of the RNA molecule is transcribed, a set of process occur to to make smaller gene transcripts.
2. The primary and secondary step occur in nucleolus.

Question 25

1. How does RNA polymerase 1 exclusively transcribe ribosomal RNA genes?
2. What is the presentation of rDNA genes?
3. Where are rRNA promoters located?
4. What are the rRNA transcription factors?
5. What is special about SL1?

Answer 25

1. rRNA is initially transcribed as a 45S transcript that is processed by Rnases to provide 28S, 18S, and 5.8S rRNAs.
2. rDNA genes are present in tandem repeats (roughly 2000 copies of 5S and 200 copies of the others).
3. rRNA promoters are located 150 bp upstream of the transcriptional start site.
4. The transcription factors: upstream binding factor (UBF) and selectivity factor one (SL1) bind the promoter and recruit RNA polymerase 1.
5. TBP is one of four subunits that make up SL1, despite the lack of a TATA box in rRNA promoters.

Question 26

What is special about the RNA polymerase 3 exclusive transcription of tRNA, 5S rRNA, and U6 snRNA?

Answer 26

The 5S rRNA and tRNA promoters are downstream of the transcriptional start site, thus the binding sites for the TFIII3s that are necessary for binding RNA polymerase 3 are located 3’ (downstream or behind) of the site where the RNApol3 itself binds and initiates transcription.
(Remember: tRNA, 5SrRNA, and U6 snRNA all use different combinations of TFIIIs)

Question 27

1. How does eukaryotic rRNA processing compare to that of prokaryotes?
2. What other processes occur in eukaryotic rRNA processing?

Answer 27

1. rRNA processing is similar in prokaryotes and eukaryotes. Each have a pre-rRNA that is transcribed and contains multiple rRNA genes. Prokaryotes undergo two cleavages to reach final products.
   Eukaryotic rRNA processing occurs in the nucleolus, where the pre-rRNA contains 18S, 5.8S and 28S. The first cleavage separates 18S from 5.8 and 28S. The second cleavage results in mature 18S rRNA and Hydrogen bonding of 5.8S to 28S rRNA.
2. In addition: Methyl groups are added to 10 bases and 100 riboses. 100 uridines are converted to pseudouridines then ribose sugar residues are added to certain bases from the conversion.

Question 28

What is the function of snoRNA and snoRNPs in eukaryotic rRNA processing?

Answer 28

Small nucleolar RNAs (snoRNAs) complex with proteins (via H bonding) to form small nucleolar ribonucleoparticles (snoRNPs).
These snoRNPs participate in the cleavage of rRNA processing in a similar way to how snRNAs participate in intron/exon splicing.
snoRNAs also function as guides for pre-rRNA base modifications (as well as base modifications for other types of RNA).

Question 29

How is eukaryotic tRNA processed?

In what 2 ways is RNase P special?

Answer 29

1. tRNAs are initially transcribed as pre-tRNAs that are folded into a functional model with sometimes multiple tRNAs located within the same pre-tRNA transcript.
2. RNAse P is an RNA and ribonucleoprotein complex but the RNA is active without the protein, making RNAse P a ribozyme. & A conventional RNAse cleaves at the 3’ end of tRNAs, but RNAse P cleaves the 5’ end of the pre-tRNAs.

Question 30

How is the 3’ end of eukaryotic tRNA processed?

Answer 30

The 3’ ends of many tRNAs are processed by addition of a CCA repeat (in other tRNAs, this is encoded) that is the site of amino acid attachment.

Question 31

What is the difference between Thymine in DNA and Uracil in RNA?

Answer 31

Thymine has a methyl group (CH3) on the 5’ Carbon of the pyrimidine ring.

Question 32

What additional processing do bases of the mature tRNA undergo?

Answer 32

Approximately 10% of all bases in mature tRNAs are modified at certain positions in the tRNA 3-stem loop structure. Modifications change base pairing properties and stabilize hairpin loops. Some tRNA and rRNA genes also have introns that are removed by splicing.