Exam 2: Eukaryotic Transcription

Question 1

Key difference between prokaryotic and eukaryotic transcription and translation

Answer 1

Prokaryotic transcription and translation can occur simultaneously, while eukaryotic transcription and translation are separated by time and space (inside of and outside of the nucleus, respectively)

Question 2

Key difference between RNA polymerase in prokaryotes and eukaryotes

Answer 2

In prokaryotes, RNA polymerase is one enzyme with four separate subunits, while in eukaryotes RNA polymerase is composed of three enzymes: pol I (rRNA), pol II (mRNA), pol III (tRNA, etc), all have many subunits, some of which are shared amongst all polymerases, some of which are unique to a single enzyme. Regardless of distribution, there are analogous subunits between pro- and eukaryotes.

Question 3

Key difference between promoter regions in prokaryotes and eukaryotes

Answer 3

In prokaryotes, the promoter is comprised of the -35 and -10 (Pribnow) boxes. In eukaryotes, the main conserved promoter is the TATA box (analogous to Pribnow box) plus a complex set of additional DNA elements.

Question 4

Key difference between proteins required for initiation in prokaryotes and eukaryotes

Answer 4

In prokaryotes, the σ (sigma) factor is required. In eukaryotes, many basal and additional factors are required.

Question 5

Key difference between RNA post-processing in prokaryotes and eukaryotes

Answer 5

In prokaryotes, no processing is performed (translation may take place simultaneously with transcription, shown), in eukaryotes significant post processing is performed as the mRNA must be transported outside the nucleus to be translated. Capping, polyadenylation and splicing of introns may occur.

Question 6

If you see an enzyme named RNA polymerase II, what indication tells you whether it is prokaryotic or eukaryotic in origin?

Answer 6

RNA polymerase II

The numbering indicates it is one of the three polymerases found in eukaryotes. Prokaryotes only have one RNA polymerase and thus it is not numbered.

Question 7

RNA polymerase I

Answer 7

Located in the nucleoli. It is responsible for the synthesis of the precursors of most rRNAs.

Question 8

RNA polymerase II

Answer 8

Located in the nucleoplasm and catalyzes the synthesis of the mRNA precursors for all protein-coding genes. RNA Pol II-transcribed pre-mRNAs are processed through cap addition, poly(A) tail addition and splicing

Question 9

RNA polymerase III

Answer 9

Located in the nucleoplasm. It is responsible for the synthesis of the precursors of 5S rRNA, tRNAs and other small nuclear and cytosolic RNAs.

Question 10

Number of subunits of eukaryotic RNA polymerases?

Answer 10

Each RNA polymerase has 12 or more different subunits. The largest two subunits are similar to each other and to the 􏰂􏰁β and 􏰂β’ subunits of E. coli RNA polymerase. Other subunits in each enzyme have homology to the 􏰃α subunit of the E. coli enzyme. Five additional subunits are common to all three polymerases, and others are polymerase specific.

Question 11

Similarities and differences between prokaryotic and eukaryotic RNA polymerases

Answer 11

Like bacterial RNA polymerases, each of the eukaryotic enzymes catalyzes transcription in a 5􏰁’ -> 3􏰁’ direction and synthesizes RNA complementary to the antisense template strand. The reaction requires the precursor nucleotides ATP, GTP, CTP and UTP (NTPs) and does not require a primer for transcription initiation. The purified eukaryotic RNA polymerases, unlike the purified bacterial enzymes, require the presence of additional initiation proteins before they are able to bind to promoters and initiate transcription.

Question 12

CTD of RNA polymerase II

Answer 12

The largest subunit of RNA polymerase II has a seven amino acid repeat at the C terminus called the carboxy-terminal domain (CTD). This sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser (YSPTSPS), is repeated 52 times in the mouse RNA polymerase II and is subject to phosphorylation. The CTD has been shown to be an important target for differential activation of transcription elongation and enhances capping and splicing.

Question 13

What can be used to distinguish the activities of the different RNA polymerases in eukaryotes?

Answer 13

Each RNA polymerase has a different sensitivity to the fungal toxin α􏰃-amanitin and this can be used to distinguish their activities.

● RNA polymerase I (RNA Pol I) transcribes most rRNA genes. It is located in the nucleoli and is insensitive to 􏰃α-amanitin

● RNA polymerase II (RNA Pol II) transcribes all protein-coding genes and some small nuclear RNA (snRNA) genes. It is located in the nucleoplasm and is very sensitive to 􏰃α-amanitin.

● RNA polymerase III (RNA Pol III) transcribes the genes for tRNA, 5S rRNA, U6 snRNA and certain other small RNAs. It is located in the nucleoplasm and is moderately sensitive to α􏰃-amanitin.

Question 14

RNA polymerase II promoters

Answer 14

Many RNA Pol II promoters contain a sequence called a TATA box which is situated 25–30 bp upstream from the start site. Other genes contain an initiator element which overlaps the start site. These elements are required for basal transcription complex formation and transcription initiation.

Question 15

RNA polymerase II enhancers

Answer 15

Enhancers are sequence elements which can activate transcription from thousands of base pairs upstream or downstream. They may be tissue-specific or ubiquitous in their activity and contain a variety of sequence motifs. There is a continuous spectrum of regulatory sequence elements which span from the extreme long-range enhancer elements to the short-range promoter elements.

Question 16

RNA polymerase II upstream regulatory elements

Answer 16

Elements within the 100–200 bp upstream from the promoter are generally required for efficient transcription. Examples include the SP1 and CCAAT boxes.

Question 17

Promoters and TATA box

Answer 17

Many eukaryotic promoters contain a sequence called the TATA box around 25–35 bp upstream from the start site of transcription (Fig. 1). It has the 7 bp consensus sequence 5􏰁’-TATA(A/T)A(A/T)-3􏰁’ although it is now known that the protein which binds to the TATA box, TBP, binds to an 8 bp sequence that includes an additional downstream base pair, whose identity is not important. The TATA box acts in a similar way to an E. coli promoter –10 sequence to position the RNA Pol II for correct transcription initiation. While the sequence around the TATA box is critical, the sequence between the TATA box and the transcription start site is not critical. However, the spacing between the TATA box and the start site is important. Around 50% of the time, the start site of transcription is an adenine residue.

Question 18

What is critical about the TATA box relative to the gene?

Answer 18

The sequence of the TATA box and its length from the start site of the gene

Question 19

Core promoter

Answer 19

The core promoter is relatively short, it consists of the TATA box and transcriptional start site

The core promoter by itself produces a low level of transcription, termed basal transcription

Question 20

Basal transcription

Answer 20

a very low level of transcription always on

Question 21

RNA polymerase II basal transcription factors

Answer 21

A series of nuclear transcription factors are required for basal transcription initiation from RNA Pol II promoter sequences in vitro. These multisubunit factors are named transcription factor TFIIA, TFIIB, etc. They have been shown to assemble on basal promoters in a specific order (D, A, B, F w/ RNA pol II, then E/H/J) and they may be subject to multiple levels of regulation.

Question 22

TFIID

Answer 22

In promoters containing a TATA box, the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element. The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex. TFIID is a multiprotein complex in which only one polypeptide, TATA-binding protein (TBP) binds to the TATA box. The complex also contains other polypeptides known as TBP-associated factors (TAFIIs). It seems that in mammalian cells, TBP binds to the TATA box and is then joined by at least eight TAFIIs to form TFIID

Question 23

TBP

Answer 23

TBP (TATA-binding protein) is a transcription factor required for transcription initiation by all three RNA polymerases. It has a saddle structure which binds to the minor groove of the DNA at the TATA box, unwinding the DNA and introducing a 45° bend. All eukaryotic TBPs analyzed have very highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription. The function of the less conserved N-terminal domain is therefore not fully understood.

Question 24

TBP introduces what changes in DNA structure?

Answer 24

1. Two bends
2. A region of unwinding between them