Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells. Unlike prokaryotic RNA polymerase that initiates the transcription of all different types of RNA, RNA polymerase in eukaryotes (including humans) comes in three variations, each translating a different type of gene. A eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures. The complexity of the eukaryotic genome necessitates a great variety and complexity of gene expression control.
Eukaryotic transcription proceeds in three sequential stages: initiation, elongation, and termination.
The RNAs transcribed serve diverse functions. For example, structural components of the ribosome are transcribed by RNA polymerase I. Protein coding genes are transcribed by RNA polymerase II into messenger RNAs (mRNAs) that carry the information from DNA to the site of protein synthesis. More abundantly made are the so-called non-coding RNAs account for the large majority of the transcriptional output of a cell. These non-coding RNAs perform a variety of important cellular functions.
RNA polymerase
Eukaryotes have three nuclear RNA polymerases, each with distinct roles and properties.
Name Location Product RNA Polymerase I (Pol I, Pol A) nucleolus larger ribosomal RNA (rRNA) (28S, 18S, 5.8S) RNA Polymerase II (Pol II, Pol B) nucleus messenger RNA (mRNA), most small nuclear RNAs (snRNAs), small interfering RNA (siRNAs) and microRNA (miRNA). RNA Polymerase III (Pol III, Pol C) nucleus (and possibly the nucleolus-nucleoplasm interface) transfer RNA (tRNA), other small RNAs (including the small 5S ribosomal RNA (5s rRNA), snRNA U6, signal recognition particle RNA (SRP RNA) and other stable short RNAs
RNA polymerase I (Pol I) catalyses the transcription of all rRNA genes except 5S. These rRNA genes are organised into a single transcriptional unit and are transcribed into a continuous transcript. This precursor is then processed into three rRNAs: 18S, 5.8S, and 28S. The transcription of rRNA genes takes place in a specialised structure of the nucleus called the nucleolus, where the transcribed rRNAs are combined with proteins to form ribosomes.
RNA polymerase II (Pol II) is responsible for the transcription of all mRNAs, some snRNAs, siRNAs, and all miRNAs. Many Pol II transcripts exist transiently as single strand precursor RNAs (pre-RNAs) that are further processed to generate mature RNAs. For example, precursor mRNAs (pre-mRNAs) are extensively processed before exiting into the cytoplasm through the nuclear pore for protein translation.
RNA polymerase III (Pol III) transcribes small non-coding RNAs, including tRNAs, 5S rRNA, U6 snRNA, SRP RNA, and other stable short RNAs such as ribonuclease P RNA.
RNA Polymerases I, II, and III contain 14, 12, and 17 subunits, respectively. All three eukaryotic polymerases have five core subunits that exhibit homology with the β, β', αI, αII, and ω subunits of E. coli RNA polymerase. An identical ω-like subunit (RBP6) is used by all three eukaryotic polymerases, while the same α-like subunits are used by Pol I and III. The three eukaryotic polymerases share four other common subunits among themselves. The remaining subunits are unique to each RNA polymerase. The additional subunits found in Pol I and Pol III relative to Pol II, are homologous to Pol II transcription factors.
Crystal structures of RNA polymerases I and II provide an opportunity to understand the interactions among the subunits and the molecular mechanism of eukaryotic transcription in atomic detail.
The carboxyl terminal domain (CTD) of RPB1, the largest subunit of RNA polymerase II, plays an important role in bringing together the machinery necessary for the synthesis and processing of Pol II transcripts. Long and structurally disordered, the CTD contains multiple repeats of heptapeptide sequence YSPTSPS that are subject to phosphorylation and other posttranslational modifications during the transcription cycle. These modifications and their regulation constitute the operational code for the CTD to control transcription initiation, elongation and termination and to couple transcription and RNA processing.
Initiation
The initiation of gene transcription in eukaryotes occurs in specific steps. First, an RNA polymerase along with general transcription factors binds to the promoter region of the gene to form a closed complex called the preinitiation complex. The subsequent transition of the complex from the closed state to the open state results in the melting or separation of the two DNA strands and the positioning of the template strand to the active site of the RNA polymerase. Without the need of a primer, RNA polymerase can initiate the synthesis of a new RNA chain using the template DNA strand to guide ribonucleotide selection and polymerization chemistry. However, many of the initiated syntheses are aborted before the transcripts reach a significant length (~10 nucleotides). During these abortive cycles, the polymerase keeps making and releasing short transcripts until it is able to produce a transcript that surpasses ten nucleotides in length. Once this threshold is attained, RNA polymerase passes the promoter and transcription proceeds to the elongation phase.
Eukaryotic promoters and general transcription factors Pol II-transcribed genes contain a region in the immediate vicinity of the transcription start site (TSS) that binds and positions the preinitiation complex. This region is called the core promoter because of its essential role in transcription initiation. Different classes of sequence elements are found in the promoters. For example, the TATA box is the highly conserved DNA recognition sequence for the TATA box binding protein, TBP, whose binding initiates transcription complex assembly at many genes.
Eukaryotic genes also contain regulatory sequences beyond the core promoter. These cis-acting control elements bind transcriptional activators or repressors to increase or decrease transcription from the core promoter. Well-characterized regulatory elements include enhancers, silencers, and insulators. These regulatory sequences can be spread over a large genomic distance, sometimes located hundreds of kilobases from the core promoters.
General transcription factors are a group of proteins involved in transcription initiation and regulation. These factors typically have DNA-binding domains that bind specific sequence elements of the core promoter and help recruit RNA polymerase to the transcriptional start site. General transcription factors for RNA polymerase II include TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH.
Assembly of preinitiation complex The transcription, a complete set of general transcription factors and RNA polymerase need to be assembled at the core promoter to form the ~2.5 million Dalton preinitiation complex. For example, for promoters that contain a TATA box near the TSS, the recognition of TATA box by the TBP subunit of TFIID initiates the assembly of a transcription complex. The next proteins to enter are TFIIA and TFIIB, which stabilize the DNA-TFIID complex and recruit Pol II in association with TFIIF and additional transcription factors. TFIIB serves as the bridge between the TATA-bound TBP and the RNA polymerase. It also helps to place the active centre of the polymerase in the correct position to initiate transcription. One of the last transcription factors to be recruited to the preinitiation complex is TFIIH, which plays an important role in promoter melting and escape.
Promoter melting and open complex formation For pol II-transcribed genes, and unlike bacterial RNA polymerase, promoter melting requires hydrolysis of ATP and is mediated by TFIIH. TFIIH is a ten-subunit protein, including both ATPase and protein kinase activities. While the upstream promoter DNA is held in a fixed position by TFIID, TFIIH pulls downstream double-stranded DNA into the cleft of the polymerase, driving the separation of DNA strands and the transition of the preinitiation complex from the closed to open state. TFIIB aids in open complex formation by binding the melted DNA and stabilizing the transcription bubble.
Abortive initiation
Once the initiation complex is open, the first ribonucleotide is brought into the active site to initiate the polymerization reaction in the absence of a primer. This generates a nascent RNA chain that forms a hetero-duplex with the template DNA strand. However, before entering the elongation phase, polymerase may terminate prematurely and release a short, truncated transcript. This process is called abortive initiation. Many cycles of abortive initiation may occur before the transcript grows to sufficient length to promote polymerase escape from the promoter. Throughout abortive initiation cycles, RNA polymerase remains bound to the promoter and pulls downstream DNA into its catalytic cleft in a scrunching-kind of motion.
Promoter escape When a transcript attains the threshold length of ten nucleotides, it enters the RNA exit channel. The polymerase breaks its interactions with the promoter elements and any regulatory proteins associated with the initiation complex that it no longer needs. Promoter escape in eukaryotes requires ATP hydrolysis and, in the case of Pol II-phosphorylation of the CTD. Meanwhile, the transcription bubble collapses down to 12-14 nucleotides, providing kinetic energy required for the escape.
Elongation After escaping the promoter and shedding most of the transcription factors for initiation, the polymerase acquires new factors for the next phase of transcription: elongation. Transcription elongation is a processive process. Double stranded DNA that enters from the front of the enzyme is unzipped to avail the template strand for RNA synthesis. For every DNA base pair separated by the advancing polymerase, one hybrid RNA:DNA base pair is immediately formed. DNA strands and nascent RNA chain exit from separate channels; the two DNA strands reunite at the trailing end of the transcription bubble while the single strand RNA emerges alone.
Elongation factors Among the proteins recruited to polymerase are elongation factors, thus called because they stimulate transcription elongation. There are different classes of elongation factors. Some factors can increase the overall rate of transcribing, some can help the polymerase through transient pausing sites, and some can assist the polymerase to transcribe through chromatin. One of the elongation factors, P-TEFb, is particularly important. P-TEFb phosphorylates the second residue (Ser-2) of the CTD repeats (YSPTSPS) of the bound Pol II. P-TEFb also phosphorylates and activates SPT5 and TAT-SF1. SPT5 is a universal transcription factor that helps recruit 5'-capping enzyme to Pol II with a CTD phosphorylated at Ser-5. TAF-SF1 recruits components of the RNA splicing machinery to the Ser-2 phosphorylated CTD. P-TEFb also helps suppress transient pausing of polymerase when it encounters certain sequences immediately following initiation.
Transcription fidelity