The prokaryotic mRNA is usually polycistronic meaning that a single primary transcript will code for several polypeptides.
The term cistron refers to a sequence which corresponds to 1 polypeptide chain plus the start and stop signals for translation.
Prokaryotic mRNA has a very short half-life (within just a few minutes the RNA is degraded by ribonucleases-RNAses)
Therefore, it is essential that translation [ protein synthesis ] be closely coupled with transcription. In prokaryotes, the ribosome can engage the mRNA before it has been completely transcribed.
Prokaryotic rRNA and tRNAs are formed by extensive processing of a long primary transcript. A single transcript may contain the sequence for several tRNAs or rRNAs or both.
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.
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 NTPs
----- 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:
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)
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.
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.