Transcription in Eukaryotes

1. The process is more complex, partially due to the nucleosomal structure of the DNA.
2. 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.
3. 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.
4. 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]
5. The primary transcript must be modified by the addition of a 5'CAP and a poly(A) tail.
6. The mature mRNA is 1/10 the size of the primary transcript due to the removal of introns by RNA splicing.
7. Eukaryotic mRNA is monocistronic

• 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.

• In the mitochondrion

-all RNAs: transcribed by a single RNA polymerase
•  In the chloroplast

-all RNAs: transcribed by a single RNA polymerase

The single kind of RNA polymerase in mitochondria and in chloroplasts is very similar to the RNA polymerase found in prokaryotes (remember that mitochondria and chloroplasts are thought to have developed from symbiontic prokaryotes). We will not discuss transcription in these organelles.
 

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

 
Using in vitro experiments specific proteins involved in transcription (called transcription factors) have been identified:
 

 2 classes:
basal transcription factors
specific transcription factors

 
Basal transcription factors necessary for transcription from all polymerase II promoters

 Specific transcription factors additionally necessary for transcription of a specific gene or a group of genes (specific regulation!)

There at least 5 basal transcription factors, [ still under investigation]
(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 (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 (see figure in text)

 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 known:
2 subunits exhibit helicase activity
1 subunit exhibits 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.

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.

 
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.
• Polyadenylation must also occur. In animal cells, the consensus sequence:

A A U A A A is a signal sequence for polyadenylation. An endonuclease cleaves the primary transcript near this site, and poly(A) polymerase adds the poly(A) tail to the 3' end of the cut. Two hundred or more As may be added!
• 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.

 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

 Transcribes only tRNA, 5S rRNA and some small RNAs.

 Promoter lies downstream of transcription start site within the transcribed region! (Fig. 6.18)

 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.

 Quiz

Quiz 2