Would you expect the bacteria to transcribe the gene? The mouse genome includes one gene and two pseudogenes for cytoplasmic thymidine kinase. Pseudogenes are genes that have lost their protein-coding ability or are no longer expressed by the cell. These pseudogenes are copied from mRNA and incorporated into the chromosome. This sequence is essential and is involved in binding transcription factors. The complexity of eukaryotic transcription does not end with the polymerases and promoters.
An army of basal transcription factors, enhancers, and silencers also help to regulate the frequency with which pre-mRNA is synthesized from a gene.
Enhancers and silencers affect the efficiency of transcription but are not necessary for transcription to proceed. Basal transcription factors are crucial in the formation of a preinitiation complex on the DNA template that subsequently recruits RNA polymerase II for transcription initiation. The transcription factors systematically fall into place on the DNA template, with each one further stabilizing the preinitiation complex and contributing to the recruitment of RNA polymerase II. Eukaryotic transcription is a tightly regulated process that requires a variety of proteins to interact with each other and with the DNA strand.
Although the process of transcription in eukaryotes involves a greater metabolic investment than in prokaryotes, it ensures that the cell transcribes precisely the pre-mRNAs that it needs for protein synthesis. The evolution of genes may be a familiar concept. Mutations can occur in genes during DNA replication, and the result may or may not be beneficial to the cell. By altering an enzyme, structural protein, or some other factor, the process of mutation can transform functions or physical features.
However, eukaryotic promoters and other gene regulatory sequences may evolve as well. For instance, consider a gene that, over many generations, becomes more valuable to the cell. We refer the reader to several excellent recent reviews that have dealt with aspects of transcription termination Rosonina et al. In addition, the cleavage complex contains CFI cleavage factor I , a dimeric protein implicated in the regulation of poly A site selection Ruegsegger et al.
The Paf1 complex Paf1C , which was first identified in yeast as an elongation factor and plays a role in transcription-associated chromatin modification Krogan et al. It is notable that one Paf1C subunit, Cdc73 or parafibromin, has been identified as a tumor suppressor protein, and the Paf1 protein is overexpressed in many cancers Chaudhary et al. Although not well studied, this RNA surveillance function is likely conserved in humans since homologs of the TRAMP and exosome complexes are present in mammals.
The CTD displays different phosphorylation patterns during the transcription cycle Phatnani and Greenleaf ; Egloff and Murphy Phosphorylation of Ser5 by the cyclin-dependant kinase CDK7, a subunit of the general transcription factor TFIIH, occurs at the initiation step of transcription to facilitate promoter release and recruitment of capping factors Komarnitsky et al.
For instance, in yeast, Ser2 phosphorylation of the CTD potentiates interaction with the essential polyadenylation factor Pcf11 de Vries et al. Interestingly, a genomic analysis employing tilling arrays in mammalian cells showed that H3-K36 methylation often decreases near the poly A site during or prior to RNAPII release, leading to the intriguing possibility that H3-K36 methylation plays a role in transcription termination Lian et al.
Differential CTD phosphorylation patterns throughout genes appear to coordinate the different steps of transcription, and dephosphorylation also contributes to this. Ssu72 might then be involved in coupling transcription termination and reinitiation via gene looping see below. CTD phosphorylation seems to modify CTD function by orchestrating the recruitment of specific factors. It is an intriguing possibility that changes in CTD phosphorylation contribute to termination.
However, as we shall see an emerging view is that the termination mechanism more likely reflects a combination of both models Fig. Poly A site recognition leads to changes in the EC. At the same time, other factors, such as Xrn2, are recruited to the EC. Involvement of chromatin remodeling factors and pausing sequences and factors are depicted.
Rat1 and its cofactor Rai1 see Xiang et al. However, depletion of Rat1 does not affect cleavage at the poly A site Kim et al. Depletion of Rat1 inhibits but does not abolish termination Kim et al. Consistent with this, the abundant and mostly cytoplasmic exonuclease Xrn1, which is similar to Rat1, is able to complement the exonuclease activity of a Rat1-deficient strain when targeted to the nucleus, but unable to complement the termination defect Luo et al.
Moreover, a defect in termination in a RAT1 mutant strain Kim et al. The above results suggest that another activity, perhaps associated with Rat1, is required to efficiently dismantle the EC. Indeed, Kawauchi et al. They suggested that Sen1 might expose the downstream RNA product generated after cleavage to facilitate its degradation by Rat1. Another possibility is that it could function at the site of termination to help disrupt the ternary complex.
Sen1 is a large, low-abundance nuclear protein that interacts with the CTD Ursic et al. Interestingly, mutations in SETX lead to two neurological disorders, an autosomal dominant form, ALS4 amyotrophic lateral sclerosis type 4 Blair et al.
The Rat1 homolog in humans is Xrn2, and its function in termination appears to be conserved. West et al. Kaneko et al. However, in both cases, poly A site recognition, which occurs rapidly, is essential for termination. These data reinforce the idea that transcription termination is a combination of both the allosteric and torpedo models Fig.
This sequence is a potential binding site for the transcription factor MAZ and promotes poly A -dependant termination. Additionally, Gromak et al. Interestingly, the strength of the poly A site correlates with efficient pausing-dependent termination Plant et al.
Other cis -acting protein-binding sites have been shown to function in termination. Another sequence, a CCAAT-box in the adenovirus late promoter is necessary for termination of the upstream gene Connelly and Manley However, whether or not it is required at every gene remains unclear. Transcription termination of protein-coding genes requires recognition of the polyadenylation signal.
The yeast mRNA-binding protein Npl3 is thought to compete for mRNA binding with cleavage and polyadenylation factors, thereby preventing termination until transcription of the poly A site Bucheli and Buratowski ; Bucheli et al. Pcf11 activity may be particularly important for transcription termination. In Drosophila , depletion of Pcf11 causes transcriptional read-through, and Drosophila Pcf11 can also dismantle the EC in vitro Zhang and Gilmour Zhang et al. In contrast to the previous experiments, this result argues that Pcf11 function in cleavage is necessary for efficient termination.
These experiments together suggest that Pcf11 may also facilitate Xrn2-mediated degradation of the downstream RNA and efficient transcription termination, but the mechanism remains unclear. This has been observed in yeast Bentley ; Calvo and Manley ; He et al. One scenario is that this facilitates recruitment of polyadenylation factors to active genes, and that these factors travel with the EC during transcription e. Alternatively, studies in yeast suggest that gene looping brings promoter and terminator regions together during early stages of transcription, facilitating RNAPII reinitiation O'Sullivan et al.
Additionally, Perkins et al. Cooperation between the splicing machinery and termination might also take place. An intriguing study in C. Cui et al. Thus, further experiments will be needed to confirm the role of PTB, and SRp20, in termination, and whether this, in fact, reflects a link with splicing. Notably, West and Proudfoot showed that termination enhances gene expression by promoting more efficient pre-mRNA processing, including post-transcriptional splicing.
The ends of mature histone mRNAs consist of a stem—loop followed by a short single-strand tail. In addition to these, another factor is required for histone pre-mRNA processing. Kolev and Steitz isolated a Heat-Labile Factor HLF , which is required for histone pre-mRNA cleavage and remarkably was found to contain polyadenylation factors, including symplekin. Indeed, deletion of the stem—loop or HDE results in transcription termination read-through Chodchoy et al.
However, in vitro and in vivo experiments suggested that cleavage of histone mRNA precursors is not necessary for termination Chodchoy et al. Recently, Yang et al. However, some data show that the degradation of the DCP can be uncoupled from cleavage Walther et al. Even if the mechanism of termination of protein-coding genes is better understood now than it was a few years ago, many aspects remain to be elucidated. Why do some genes terminate closer to the poly A site than the others?
Are the downstream genes important elements in the choice of a termination site? Indeed, some data suggest that endonucleolytic cleavage at the poly A site is not a prerequisite for transcription termination Osheim et al. Is it then possible that the mechanism of transcription termination can vary among genes? This is perhaps not surprising, given precedents from prokaryotes, such as rho-independent and rho-dependent mechanisms of termination Nudler and Gottesman Although many elements and protein factors important for termination have been discovered recently, it remains difficult to draw a detailed common picture of RNAPII termination.
Medlin et al. Interestingly, phosporylation of Ser7 of the CTD is required for the recruitment of the Integrator complex and, therefore, for snRNAs transcription and processing Egloff et al.
However, inactivation of Int11 did not seem to interfere with termination of transcription. However, such a sequence was not identified around the natural terminator region of U2. These experiments indicate that snRNA transcription termination might involve gene-specific factors and mechanisms, although details remain unclear.
There is no evidence yet of an association of Xrn2 with snRNAs genes, but it is conceivable that termination occurs via degradation of the downstream product, at least in U2 termination. Termination of yeast snRNA transcription involves the same pathway used for snoRNA termination, which is discussed next. The majority of snoRNAs fall into two structurally and functionally well-defined classes. Termination at terminator I TI , the major termination site, is Nrd1-binding-dependent. After release of the precursor, Trf4 and Pap1 adenylates the pre-snoRNA that will be processed by the exosome.
Other factors shown to have a role in snoRNA termination are indicated. Kim et al. However, transcription read-through was seen in strains expressing PCF11 mutants that are not capable of binding the CTD, and Garas et al. Interestingly, as shown in mRNA termination, Steinmetz et al. The above results indicate that snoRNA genes appear to use different pathways to terminate transcription, as mutations or depletion in factors involved in mRNA termination have effects only on a subset of snoRNA genes.
Further investigations will be needed to understand what underlies the differences among these genes. Several snoRNA terminators contain multiple potential binding sites for Ndr1 and Nab3, which form a heterodimer Carroll et al. Nrd1 can also recruit the exosome complex, coupling the Nrd1 termination pathway to exosome-dependent trimming or degradation of transcripts Fig. Nedea et al. Prokaryotic transcription is much simpler than eukaryotic transcription.
For instance prokaryotes have only one RNA polymerase that carries out the complete process of transcription. Furthermore each eukaryotic polymerase carries out its necessary functions at different locations in the cell. Prokaryotic transcription is carried out in the cytoplasm, where transcription is coupled with translation 1.
This is probably due to the fact that it takes place in the cytoplasm and is subjected to nuclease degradation. Another difference between prokaryotic and eukaryotic transcription are the subunits that make up the polymerases themselves. Eukaryotic Pol II is made up of 10 subunits. Eukaryotic transcription is carried out in the nucleus of the cell by one of three RNA polymerases, depending on the RNA being transcribed, and proceeds in three sequential stages:.
Unlike the prokaryotic RNA polymerase that can bind to a DNA template on its own, eukaryotes require several other proteins, called transcription factors, to first bind to the promoter region and then help recruit the appropriate polymerase.
The completed assembly of transcription factors and RNA polymerase bind to the promoter, forming a transcription pre-initiation complex PIC. The most-extensively studied core promoter element in eukaryotes is a short DNA sequence known as a TATA box, found base pairs upstream from the start site of transcription.
However, only a low, or basal, rate of transcription is driven by the pre-initiation complex alone. Other proteins known as activators and repressors, along with any associated coactivators or corepressors, are responsible for modulating transcription rate. Activator proteins increase the transcription rate, and repressor proteins decrease the transcription rate. Transcription factors recognize the promoter, RNA polymerase II then binds and forms the transcription initiation complex.
The features of eukaryotic mRNA synthesis are markedly more complex those of prokaryotes. Instead of a single polymerase comprising five subunits, the eukaryotes have three polymerases that are each made up of 10 subunits or more. Each eukaryotic polymerase also requires a distinct set of transcription factors to bring it to the DNA template.
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