Transcription in Eukaryotes

(Lodish et al., 2000, Section 10.3, 10.6 )

Several major differences in transcription compared to prokaryotes:

The process is more complex, partially due to the nucleosomal structure of the DNA.
There are 3 different RNA polymerases in the nucleus of eukaryotic cells [termed RNA polymerases I, II, & III] plus an additional mitochondrial RNA polymerase and chloroplast RNA polymerase.
All three of the nuclear RNA polymerases do not bind directly to their promoters but to proteins [termed transcription factors] that are in turn bound to specific DNA sequences that constitute each promoter.
The mRNAs are longer lived and the processes of transcription and translation are spatially and temporally separated [i.e. transcription occurs in the nucleus, translation occurs in the cytoplasm]
The primary transcript must be modified by the addition of a 5'CAP and a poly(A) tail.
The mature mRNA is 1/10 the size of the primary transcript due to the removal of introns by RNA splicing.
Eukaryotic mRNA is monocistronic

Each RNA polymerase transcribes specific classes of RNA:

RNA polymerase I is responsible for transcribing the 5.8s rRNA, the 18s rRNA, and the 28s rRNA.
RNA polymerase II is responsible for transcribing mRNA.
RNA polymerase III is responsible for transcribing the 5s rRNA and the tRNAs.
(Fig. 10.26)
In the mitochondrion

In the chloroplast

RNA polymerase

mitochondria

chloroplasts

Experiment - Transcription of RNA Polymerase II

In the test tube (in vitro) isolated RNA polymerase II plus DNA plus NTP

----- does not lead to transcription as was previously discovered for E. coli
addition of other proteins

-----successful transcription in vitro

Conclusion: additional proteins must be present that fulfill role of the sigma subunit but are not primarily associated with RNA polymerase II

Auxillary Proteins Needed for Transcription
Using in vitro experiments specific proteins involved in transcription (called transcription factors) have been identified.
There are two classes of factors:

basal transcription factors
specific transcription factors

Basal transcription factors initiating Eukaryotic transcription (Fig. 10.50).

There at least 5 basal transcription factors, (TFIIB, TFIID, TFIIE, TFIIF, TFIIH), (TF indicates transcription factor, II indicates specificity for polymerase II)
Sequence comparisons of RNA polymerase II promoters have revealed a consensus sequence of TATAA at -25 to -30, called TATA box (Fig. 10.30) (note similarity to -10 sequence or TATAAT in prokaryotes)
A protein called TATA-binding protein (TBP) binds specifically to the TATA box. TBP is part of a large protein complex called TFIID that also contains other polypeptides called TBP-associated factors (TAFs). Transcription is initiated by TFIID specifically binding to the TATA box via its TBP subunit
The TBP subunit can then bind another transcription factor called TFIIB forming a TBP-TFIIB complex
Only now can RNA polymerase II bind to the promoter via binding to the TBP-TFIIB complex. However, it does so only in association with a third transcription factor called TFIIF.
Before transcription can really be initiated in vitro, 2 other transcription factors must bind to RNA polymerase II: TFIIE and TFIIH.
The enzyme activities exhibited by some of the subunits of TFIIH are helicase activity and protein kinase activity (phosphorylates proteins)

The helicase activity of TFIIH is thought to unwind the DNA at the start site of transcription

The protein kinase activity is thought to cause the RNA polymerase II to leave the promoter so elongation can occur. TFIIH seems to phosphorylate specific amino acid side chains in the RNA polymerase II (introduces negative charges!) and this seems to provide the force to disrupt the strong interaction between the initiation complex and the polymerase.

Not all promoters for RNA polymerase II contain a TATA box.

A second important sequence element was found in many polymerase II promoters called initiator sequence (Inr). Inr can represent the only specific sequence in a polymerase II promoter or it can occur together with a TATA box.
Inr is recognized by other subunits of the TFIID complex (the TAFs) and transcription proceeds in the same order as described before. But although in the Inr case TBP does not provide the specific binding, it is none-the-less required as part of the TFIID complex for transcription to occur and therefore seems to play a central role in initiation.

From in vitro to in vivo (in the living cell)?
Much of the transcription process by RNA polymerase II still needs to be elucidated. For example, the function of many of the basal transcription factors is not known. And the way transcription actually occurs in the living cell could still be different from the way it occurs in the test tube.

Post-transcriptional Modification to form mRNA

The primary eukaryotic transcript must be modified in several ways in order to form a mature mRNA.
First, shortly after transcription begins, a CAP is added to the 5' end of the transcript. This CAP is a 7-methyl guanosine (Fig. 4.18).
Polyadenylation must also occur. In animal cells, the consensus sequence:

Fig.4.19

Finally, all introns[intervening sequences] are removed in a process known as RNA splicing and the remaining exons are joined to yield the mature mRNA.

RNA polymerase I:

Transcribes only the 5.8S, 18S, and 28S rRNAs. Transcription occurs in nucleolus.
Transcribes them as one large 45S pre-rRNA containing one copy of each plus spacers (noncoding regions) inbetween (Fig. 6.16)
Subsequent processing yields the individual rRNAs.
Promoter lies directly upstream of initiation site. Promoter is specifically recognized by transcription factors UBF (upstream binding factor) and SL1 (selective factor 1).
A subunit of SL1 is TBP (TATA-binding protein)! (Fig. 6.17)
Discovered because yeast mutants in TBP show no transcription from promoters for RNA polymerase I, II and even III
---- central role for TBP in all nuclear transcription
But TBP not involved in specific recognition of promoter (no TATA box in RNA polymerase III promoters), instead specific recognition through other SL1 subunits

(Fig 10.69)

RNA polymerase III:

Transcribes only tRNA, 5S rRNA and some small RNAs.
Promoter lies downstream of transcription start site within the transcribed region!
5S rRNA:

TFIIIA initiates specific binding, recruits
TFIIIC, TFIIIB, and RNA polymerase III

note TBP as important subunit of TFIIIB!!
tRNA transcription slightly different as no TFIIIA involved. Instead, specific promoter recognition by TFIIIC which in turn recruits TFIIIB and polymerase III.

Activity

Quiz

References:
Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D., Darnell, J., 2000, Molecular Cell Biology, 4th Ed., W.H. Freeman and Company, NY, New York. ISBN 0-7167-3136-3.