Differences between prokaryotic and eukaryotic transcription

Eukaryotic transcription has some similarities to prokaryotic transcription, but is more complex.

Chromatin must be opened before transcription can occur

The basic principle is the same, a promoter is still used as an initiation site for transcription, but while in prokaryotes the transcription acts on a DNA template, in eukaryotes transcription acts on a chromatin template.

Chromatin changes everything and must be taken into account at every step. For example, the chromatin structure of DNA must be opened before RNA polymerase can bind to the promoter.

Figure 1: In contrast to closed chromatin, permissive chromatin is sufficiently dynamic for transcription factors to initiate sequence-specific accessibility remodelling and establish an open chromatin conformation (illustrated here for an active gene locus). Pol II, RNA polymerase II; TF, transcription factor. (https://www.nature.com/articles/s41576-018-0089-8)

More transcription factors are required for initiation

Furthermore, eukaryotic transcription requires a different subset of transcription factors. These are defined as any protein that modulates the activity of transcription, but is not itself part of the RNA polymerase.

A second major difference is that bacterial RNAP can read the DNA sequence to find and bind the promoter. A eukaryotic RNAP cannot read DNA. Instead, transcription initiation requires a large number of factors which must pre-bind to a variety of cis-acting elements before the RNAP can bind.

These are known as basal transcription factors. RNAP then binds to the basal transcription factor-DNA complex. The binding region is known as the core promoter - the region containing all the binding sites necessary for RNAP to bind and function.

There are multiple types of RNAP

While bacteria have a single type of RNAP, eukaryotes have three types of RNAP, which are each responsible for transcribing one of the major classes of genes in eukaryotes:

RNAP I transcribes rRNA (but not 5S rRNA).
RNAP II transcribes mRNA and some small RNAs.
RNAP III transcribes tRNA, 5S ribosomal RNA, and other small RNAs.

The promoters for RNAP I and II are mostly located upstream of the start point, but a large number of promoters for RNAP III are located downstream of the start point, within the transcription unit.

An example schematic of a promoter for RNAP III, transcribing a tRNA gene showing regulatory sequences downstream of the start point.

Other sequences, known as enhancers can be located much further upstream or downstream of the promoter, and control whether the promoter is expressed or not.

Figure 2: A typical gene transcribed by RNA polymerase II has a promoter that extends upstream from the site where transcription is initiated. The promoter contains several short-sequence (~10 bp) elements that bind transcription factors, dispersed over ~100 bp. An enhancer containing a more closely packed array of elements that also bind transcription factors may be located several hundred base pairs to several kilobases distant. (DNA may be coiled or otherwise rearranged so that transcription factors at the promoter and at the enhancer interact to form a large protein complex.)

Eukaryotic RNAPs consist of many subunits

RNAP I

The most prominent RNAP of the three is RNAP I, which resides in the nucleolus and is responsible for transcribing the 18S, and 28S rRNA. It accounts for most of the cellular RNA synthesis in terms of quantity.

RNAP II

RNAP II, located in the nucleoplasm (the region of the nucleus not including the nucleolus), is responsible for synthesising heterogeneous nuclear RNA (hnRNA).

hnRNA is the precursor to most mRNA, and a lot more, with the classical definition being that hnRNA is all RNA which is not rRNA or tRNA.

RNAP III

RNAP III is a minor enzyme in terms of activity but is responsible for producing a collection of stable, essential RNAs. This nucleoplasmic enzyme synthesises the 5S rRNAs, tRNAs, and other small RNAs that constitute over a quarter of cytoplasmic RNA.

Figure 3: An overview of the relative abundance of the different types of RNA in eukaryotes.

Localisation of RNAP I II and III in the nucleus.

Eukaryotic RNAPs are large complexes

All RNAPs are large complexes of many subunits. RNAP II, for example, is approximately 500kD and comprised of 12 or so subunits, while in yeast RNAP I has 14 subunits and RNAP III has 17.

The two largest subunits are homologous to the $\beta$ and $\beta'$ subunits of prokaryotic RNAP.

Five of the remaining subunits (in yeast, Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) are common to all the RNAPs (I, II, and III), however there is no analogous subunit in eukaryotic RNAP to that of $\sigma$ factor of prokaryotic RNAP, that function is contained within the basal transcription factors.

Figure 4: Some subunits are common to all classes of eukaryotic RNA polymerases and some are related to bacterial RNA polymerase. This drawing is a simulation of purified yeast RNA polymerase II run on an SDS gel to separate the subunits by size.

The large subunit of RNAP II

The largest subunit of RNAP II has a carboxy-terminal domain (CTD) which consists of multiple repeats of a consensus sequence of amino acids. This sequence is unique to RNAP II.

The number of repeats is important because mutations that remove more than half of them are lethal. The CTD is involved in regulating the initiation reaction, transcription elongation, and all aspects of mRNA processing, and even export of mRNA to the cytoplasm.

RNAP I’s Promoters

RNAP I transcribes only the genes for ribosomal RNA from a single type of promoter in a special region of the nucleus called the nucleolus (a small, dense region of the nucleus that is responsible for the synthesis of ribosomal RNA).

The precursor transcript includes the sequences of both the large 28S and small 18S rRNAs, which are later processed by cleavages and modifications. Ribosome assembly also occurs in the nucleolus.

There are many copies of the rRNA transcription unit, joined together by non-transcribed spacers and are organised into a cluster (Figure 5).

Figure 5: A tandem gene cluster for rRNA genes

The promoter structure is shown in Figure 6.

Figure 6: Transcription units for RNA polymerase I have a core promoter separated by ~70 bp from the upstream promoter element. Upstream binding factor (UBF) binding to the upstream promoter element (UPE) increases the ability of core-binding factor to bind to the core promoter. Core-binding factor (SL1) positions RNA polymerase I at the start point.

The structure consists of two separate regions (the promoter structure is often called bipartite):

The core promoter surrounds the start point, extending from -45 to +20, and is sufficient for transcription to initiate. It’s a GC rich region, except for a short stretch of AT rich sequence around the start point.
The upstream promoter element (UPE) is another GC rich sequence, extending from -180 to -107, and is responsible for binding the upstream binding factor (UBF).

RNAP I requires two transcription factors to recognise the promoter sequence. The factor that binds to the core promoter is SL1, a multisubunit complex containing one TATA-binding protein (TBP) and several Pol I-specific TBP-associated factors (TAFs). TBP also participates in initiation by RNAP II and III (we will not discuss the second transcription factor in this course).

SL1 is primarily responsible for the binding of RNAP I at the start point, and also responsible for transcription initiation.

The Basal Apparatus Assembles at the Promoter of RNAP II

In the cell, promoters can be found in three basic types of chromatin:

An inactive gene will be in closed chromatin.
A potentially active gene in open chromatin, bound to RNAP, is called a poised gene.
An active gene in open chromatin.

Poised genes may assemble the basal apparatus, but they cannot proceed to transcribe the gene without a second signal to initiate transcription.

Transcription initiation requires that the basal transcription factors act in a defined order that will be joined by the RNAP. Figure 7 shows the ordered assembly of the basal apparatus at the promoter of RNAP II.

Figure 7: An initiation complex assembles at promoters for RNA polymerase II by an ordered sequence of association with transcription factors.

The factors join in a fixed order. TF $_{II}$ D, which contains TBP and several TBP-associated factors (TAFs), binds the promoter first, and TF $_{II}$ B joins it. RNAP II then arrives together with TF $_{II}$ F, followed by TF $_{II}$ E and finally TF $_{II}$ H. Phosphorylation of the CTD converts the polymerase from the IIa form to the IIo form, which leaves the promoter with TF $_{II}$ F to transcribe the gene; TF $_{II}$ E and TF $_{II}$ H are released, while TBP and TF $_{II}$ B can remain at the promoter.

RNAP III’s Promoters and transcription factors

RNAP III has three types of promoters, all of which are recognised in different ways by different factors.

Two of the promoter types are internal promoters. Type 1 5S rRNA promoters contain A, intermediate, and C elements and recruit TF $_{III}$ A followed by TF $_{III}$ C. Type 2 promoters, including tRNA promoters, contain A and B boxes that bind TF $_{III}$ C directly; TF $_{III}$ C then recruits TF $_{III}$ B and Pol III (Figure 8).

Figure 8: Promoters for RNA polymerase III may consist of bipartite sequences downstream of the start point, with boxA separated from either boxC or boxB, or they may consist of separated sequences upstream of the start point (Oct, PSE, TATA).

These factors assemble on promoter elements downstream of the start point and position TF $_{III}$ B upstream of it. TF $_{III}$ B then recruits Pol III to the start point.

Figure 9: Internal type 2 pol III promoters use binding of TF $_{III}$ C to boxA and boxB sequences to recruit the positioning factor TF $_{III}$ B, which recruits RNA polymerase III.

TF $_{III}$ A and TF $_{III}$ C in type 1 promoters, and TF $_{III}$ C in type 2 promoters, act as assembly factors that assist the binding and positioning of TF $_{III}$ B. Once TF $_{III}$ B is bound, these assembly factors can be removed without affecting the initiation reaction. These transcription factors form part of the basal apparatus.

Like SL1, in RNAP I promoter assembly, TF $_{III}$ B contains the TATA-binding protein (TBP).

TF $_{III}$ B remains bound in the vicinity of the start point, and its presence is sufficient to allow RNA polymerase III to identify and bind at the start point. Thus, TF $_{III}$ B is the only true initiation factor required by RNA polymerase III.

TATA Binding Protein (TBP) is a Universal Factor

TBP is a component of the Pol I factor SL1, the Pol II factor TFIID (often itself called TBP), and the Pol III factor TF $_{III}$ B. It directly recognises TATA DNA at TATA-containing promoters, while other complex components help position the machinery at TATA-less promoters.

Figure 10: RNA polymerases are positioned at all promoters by a factor that contains TBP.

TBP binds DNA in the minor groove (this is unusual for a DNA binding protein). It forms a “saddle” like structure around the DNA, causing it to bend by approximately 80 degrees. This sharp kink in the DNA is accompanied by significant unwinding of the DNA and allows for other transcription machinery to bind.

TBP’s larger outer surface is exposed and available to contact other proteins for the recruitment of RNAP.

Figure 11: The cocrystal structure of TBP with DNA from −40 to the start point shows a bend at the TATA box that widens the narrow groove where TBP binds.

Enhancers

Until now we have considered the promoter as an isolated region responsible for binding the RNA polymerase. Eukaryotic promoters do not necessarily function alone.

In most cases, the activity of a promoter is substantially increased by the presence of an enhancer sequence located at a variable distance from the core promoter.

Some enhancers function through very long-range interactions of tens of kilobases, others function through short-range interactions and may be quite close to the promoter.

The CCAAT box is a common promoter-proximal regulatory element, often near -80, that can influence promoter strength; while often introduced as an example of an enhancer (if you read the textbook!) it is not itself an enhancer due to its location and mechanism of action.

Why are enhancers not considered part of the promoter though? This is mainly due to two characteristics:

Enhancers need not be at a fixed position, they can be upstream, downstream, and great distances away.
Enhancers can function in any orientation (i.e they can be inverted and still work).

Figure 12: An enhancer can activate a promoter from upstream or downstream locations, and its sequence can be inverted relative to the promoter.

Transcription factor types

Transcription factors (TFs) can be divided into two groups:

Activators, which are TFs that positively stimulate transcription.
Repressors, which are TFs that negatively downregulate transcription. For example, REST binds a DNA element found near many genes that are needed only in neurons, and keeps those genes silent in other cell types.

Much of a cell’s gene expression profile is determined by the balance of different transcription factors currently active in the cell.

Even at the single-gene level, the activity of a gene can be regulated by the presence of different activators or repressors. If more activators are present, the gene is transcribed more, if more repressors are present, the gene is transcribed less.

Enhancer driven transcription factors

We can further distinguish enhancer-binding transcription factors and co-activators:

True activators are those that function by both binding specific DNA sites and making contact with the basal machinery at the promoter.
Architectural transcription factors: Their sole function is to change the structure of DNA, typically to bend it. This can then facilitate the bringing together of two transcription factors separated by short distances in order to initiate transcription.
Co-activators are factors recruited by DNA-bound activators. They stimulate transcription through mechanisms that include chromatin modification and direct effects on pre-initiation-complex assembly.

See the following video for a high level overview of transcription, including the role of enhancers: https://www.youtube.com/watch?v=SMtWvDbfHLo

Gene Expression is Associated with Demethylation

Methylation of DNA is a key epigenetic (meaning a change that does not alter the DNA sequence itself, but is often heritable) regulatory event that can influence the activity of a promoter. At promoters silenced by DNA methylation, methylation can prevent transcription even if the specific transcription factors for that promoter are present (Figure 13).

Figure 13: A simplified model showing how promoter methylation can be associated with reduced transcription and protein expression. This methylated-off/unmethylated-on pattern is not universal.

The effect of methylation is well characterised at promoters for RNAP I and RNAP II.

While DNA methylation is a reversible process, it can be stably maintained over many cell divisions. Methylation also occurs in a particular epigenetic phenomenon known as imprinting. In sexual reproduction, most methylation information from the sperm or oocyte is erased. However, imprinted genes keep their methylation patterns. This occurs in sex-specific patterns in the sperm or oocyte, which results in maternal and paternal alleles being differentially expressed in the offspring.

In eukaryotes, and especially mammals, methylation typically occurs on carbon 5 of cytosine (creating 5-methylcytosine) at CG doublets. These CG dinucleotides are called CpG sites; CpG islands are longer regions enriched in CpG sites. These sites are methylated by enzymes called DNA methyltransferases (DNMTs):

A maintenance methyltransferase acts on DNA that is already methylated on one strand of a CG doublet after replication.
A de novo methyltransferase can methylate a previously unmethylated CG site.

CpG islands are regions of DNA that are rich in CG doublets (the term CpG is used to explicitly refer to Cytosine-Phosphate-Guanine, i.e the DNA sequence on a single strand, as opposed to a CG base pair). They are major regulatory units and around 50% of CpG islands are located in gene promoter regions, while another 25% lie in gene bodies. Around 60-70% of human genes have a CpG island in their promoter region.

CpG island in a promoter region in mammalian genes

At promoters silenced by DNA methylation, loss of promoter methylation can permit activation, but promoter demethylation is not universally required for transcription. While the process is complex, enzymes known as TET enzymes are key players. They oxidise the methyl group of 5-methylcytosine in steps, initiating a repair pathway that replaces the modified base with an unmethylated cytosine.

Generally, a gene that is actively being transcribed is described as being undermethylated, particularly at its 5’ promoter region. Conversely, a lack of gene expression is associated with methylation in this promoter region. It’s important to note, however, that the body of an active gene is often methylated. At promoters silenced by DNA methylation, demethylation can contribute to activation.

Indirect evidence for this relationship comes from experiments using the drug 5-azacytidine. This drug is incorporated into DNA but cannot be methylated. This leads to demethylation of the DNA during replication and can induce changes in cell differentiation, such as activating muscle-specific genes in non-muscle cells. This supports the idea that demethylation can permit activation of genes silenced by DNA methylation.