Chapter 13: Mechanisms of Transcription

The "scrunching" model proposes that DNA downstream from the stationary, promoter bound polymerase is unwound and pulled into the enzyme. This is the model that is currently accepted.

During initial transcription, RNA polymerase produces and releases short RNA transcripts of <10 nucleotides (abortive synthesis) before escaping the promoter, entering elongation phase, and synthesizing proper transcript. How does this occur?

TFIIB

General transcription factor that enters the preinitiation complex after TBP forming a ternary complex of TFIIB-TBP-DNA. The asymmetric binding of this protein to the TBP- DNA complex accounts for the asymmetry in the rest of the assembly of the preinitiation complex and the unidirectional transcription that results. This protein also contacts Pol II in the preinitiation complex, bridging the TATA bound TBP and polymerase. Structural studies also suggest that segments of this protein insert into the RNA exit Channel and active center cleft of Pol II in a manner that is analogous to the sigma factor 3/4 linker in bacteria. These regions of the protein (called the linker and reader) aid in the open complex formation by stabilizing the melted DNA until the RNA:DNA hybrid takes over that role.

expressed a lot

Genes with strong consensus sequences are typically

Instead, these promoters have an "extended -10 element". This comprises a standard -10 element with an additional short sequence element at its upstream end. Extra contacts between the polymerase and this sequence compensate for the absense of a -35 element.

In a class of sigma 70 factor promoters that lack a -35 region, what is in its place?

TBP

In addition to RNA Pol II, eukaryotes have two other RNA polymerases: Pol I and Pol III. These enzymes are related to Pol II and even share several subunits, but they initiate transcription from distinct promoters and transcribe distinct genes. Those genes encode specialized RNAs rather than proteins. Each of these enzymes also works with its own unique set of general transcription factors. However, —— is universal, and it is involved in initiating transcription by Pol I and III, as well as Pol II.

allosteric model of termination

Model of termination in which termination depends on a conformational change in the elongating polymerase that reduces the processivity of the enzyme leading to a spontaneous termination soon afterward. This conformational change would be linked to polyadenylation and could be triggered by the transfer of the 3'processing enzymes from the CTD tail of polymerase to the RNA or by the subsequent binding to the CTD tail of other factors that induce a conformational change.

Helix-turn-helix motif

Motif often involved in DNA binding

RNA Polymerase III

RNA polymerase in eukaryotes whose promoters are unusual in that they are typically located downstream from the transcription start site (within the coding region of the gene). Some promoters of this polymerase (those for the tRNA genes) consist of two regions called Box A and Box B separated by a short element. Others contain Box A and Box C (e.g. the 5S rRNA gene). And others contain a TATA element like those of Pol II. Just as with the other RNA polymerases, transcription by this polymerase requires transcription factors in addition to polymerase. In this case, the factors are called TFIIIB and TFIIIC for the tRNA genes and those plus TFIIIA for the 5S rRNA gene.

T7 polymerase

RNA polymerase of E. Coli phage T7 that transcribes the genes of the phage upon infection. It is more structurally related to DNA polymerases ("right hand model" with the fingers, thumb and plan representing domains arranged around a central cleft with the active site) than RNA polymerases. It has a molecular mass of 100kDa. Although it is not structurally related to RNA polymerases, it does have features functionally analogous to them. For example, it has sigma like functions that open up the RNA exit channel, allowing production of transcripts larger than 10 nucleotides.

The DNA template in vivo is packaged into chromatin, and this condition complicates binding to the promoter of polymerase and its associated factors.

Regulated transcription in vivo requires additional transcriptional regulator proteins, the mediator complex, and nucleosome modifying enzymes. Why?

—

Review how consensus sequences are found!!

core promoter

The minimal set of eukaryotic sequence elements required for accurate transcription initiation by the Pol II machinery, as needed in vitro. Typically 40-60 nucleotides long, extending either upstream or downstream from the transcription start site

Sigma 70 factor

The predominant sigma factor in E. Coli.

Two protein complexes are carried by the CTD of the polymerase as it approaches the end of the gene: CPSF (cleavage and polyadenylation specificity factor) and CTSF (cleavage stimulation factor). The sequences that, once transcribed into RNA, trigger transfer of these factors to the RNA are called poly-A signals. Once CPSF and CSTF are bound to the RNA, other proteins are recruited as well, leading initially to RNA cleavage and then polyadenylation. Polyadenylation is mediated by an enzyme called poly-A polymerase, which adds approximately 200 adenines to the RNA's 3' end produced by the cleavage. This enzyme uses ATP as a precursor and adds the nucleotides using the same chemistry as RNA polymerase (but without a template). Thus, the long tail of As is found in the RNA but not the DNA. The mature mRNA is then transported out of the nucleus.

What are the steps involved in polyadenylation?

UP element

A DNA element that binds RNA polymerase and is found in some strong promoters. It increases polymerase binding by providing an additional specific interaction between the enzyme and DNA.

discriminator

A DNA element that binds RNA polymerase and is sometimes found just downstream from the -10 element. The strength of the interaction between this element and polymerase influences the stability of the complex between the enzyme and the promoter.

mediator complex

A complex associated with the basic transcription machinery, most likely touching the CTD tail of the large polymerase subunit through one surface, while presenting other surfaces for interaction with DNA bound activators. Despite this central role in transcription activation, deletion of individual subunits of the complex often leads to loss of expression of only a small subset of genes, different for each subunit. This result reflects the fact that different activators are believed to interact with different ———— subunits to bring polymerase to different genes. In addition, this complex aids initiation by regulating the CTD kinase in TFIIH.

TFIID

A multi subunit General transcription factor that recognizes the TATA element. It is a critical factor in promoter recognition and preinitiation complex establishment.

In yeast, both DNA replication and gene transcription initiation are largely controlled by DNA sequence elements

Clicker question about DNA replication and transcription

RNA polymerase holoenzyme

Complex of RNA polymerase and associated sigma factor (initiation factor) that is only able to intitiate transcription at promoters. (Without the sigma factor, RNA polymerase can initiate transcription at any point on a DNA molecule.)

TATA element

Core promotor sequence element found 30bp upstream from the transcription start site in many Pol II promoters. This is where preinitiation complex formation begins.

A subunit of TFIIH acts as an ATP driven trans locator of double stranded DNA. This subunit binds to DNA downstream from polymerase and feeds double stranded DNA, with a right handed threading, into the cleft of the polymerase. This action drives the melting of the DNA because the upstream promotor DNA is held in a fixed position by TFIID and the rest of the GTFs.

How does TFIIH mediate promoter melting?

RNA polymerase does not immediately terminate after the RNA is cleaved and polyadenylated. Rather, it continues to move along the template, generating a second RNA molecule. The polymerase can continue transcribing for several thousand nucleotides before terminating and dissociating from the template.

How does transcription terminate generally?

consensus sequence

A sequence that reflects preferred -10 and -35 regions of a promoter separated by optimum spacing (17bp). Very few promoters have this exact sequence but must differ from it by only a few nucleotides. Promoters with sequences closer to the ———- are generally "stronger" than those that match less well.

As is bacteria, in eukaryotes a period of abortive initiation occurs before the polymerase escapes the promoter and enters the elongation phase. Promoter escape involves two steps not seen in bacteria: ATP hydrolysis needed for DNA melting and phosphorylation of the polymerase. Addition of phosphates to the Pol II tail helps polymerase shed most of the general transcription factors used for initiation, which the enzyme leaves behind as it escapes the promoter.

Describe promoter escape in eukaryotes

There are five channels into the enzyme. The NTP uptake channel allows ribonucleotides to enter the active center. The RNA exit channel allows the growing RNA chain to leave the enzyme as it is synthesized during elongation. The three remaining channels allow DNA entry and exit from the enzyme. The downstream untranscribed DNA enters the active center cleft in double stranded form through the downstream DNA channel between the pincers. Within the active center cleft, the DNA strands separate from position +3. The nontemplate strand exits the active center cleft through the non-template strand channel and travels across the surface of the enzyme. The template strand follows a path through the active center cleft and exits through the template strand channel. The double helix reforms at -11 in the upstream DNA behind the enzyme.

Describe the general structure of the RNA polymerase holoenzyme.

Overall, the shape of each enzyme resembles a crab claw. The two pincers of the crab claw are made up of predominantly of the two largest subunits of each enzyme (beta and beta' for bacteria and RPB1 and RPB2 for eukaryotes). The active site, which is made up of regions from both of these subunits, is found at the base of the pincers within a region called the "active center cleft". The active site works according to the two metal cation catalytic mechanism for nucleotide addition proposed for all types of polymerase . In this case however, the active site contains only one tightly bound Mg2+ ion and the second Mg2+ is brought in which each new nucleotide in the addition cycle and released with the pyrophosphate.

Describe the general structure of the bacterial RNA polymerase core enzyme (RNA Pol II in eukaryotes.

1. The gamma phosphate at the 5' end of the RNA is removed by an enzyme called RNA triphosphatase. 2. The enzyme guanyltransferase adds a GMP moiety to the resulting beta phosphatase. This is a two step process: first, an enzyme-GMP complex is generated from GTP with release of the beta and gamma phosphates of that GTP and then the GMP from the enzyme is transferred to the beta phosphate of the 5' end of the RNA. 3. Once this linkage is made, the newly added guanine and the purine at the original 5' end of the mRNA are further modified by the addition of methyl groups by methyltransferase. The resulting 5' cap structure subsequently recruits the ribosome to the mRNA for translation to begin.

Describe the steps involved in capping.

Whereas bacteria only have one RNA polymerase, all eukaryotes have at least three (I, II, and III). In addition, whereas bacteria require only one additional factor (sigma), several initiation factors (general transcription factors) are required for efficient and promotor specific initiation in eukaryotes. (One similarity is that the bacterial RNA polymerase and the eukaryotic RNA polymerase have a similar structure.)

Differences between transcription in bacteria and eukaryotes

5'-3'

Direction of transcription

It can only do this once it has managed to synthesize a transcript of a threshold length of 10 or more nucleotides. Once this length, the transcript cannot be accommodated within the region where it hybridizes to the DNA and must start threading into the RNA exit channel. Promotor escape is associated with the breaking of all interactions between polymerase and promotor elements and between polymerase and any regulatory proteins operating at the given promoter. In addition, the region called the 3/4 linker of the sigma factor lies in the middle of the RNA exit channel in the open complex, and for an RNA chain to be made longer than 10 nucleotides, this region of sigma must be ejected from that location. This can take multiple attempts.

How does the polymerase manage to escape the promotor and enter the elongation phase?

The DNA binding regions of sigma factor point away from the body of the enzyme, rather than being embedded. Moreover, the spacing between those regions is consistent with the distance between the DNA elements they recognize. (The space between them is the same as the space between the -10 and -35 elements.)

How is the sigma subunit positioned within the holoenzyme structure in such a way as to make feasible the recognition of various promoter elements?

TFIIH

Largest and most complex general transcription factor that has 10 subunits and a molecular mass comparable to Pol II. This protein controls the ATP- dependent transition of the preinitiation complex to the open complex. Within the protein are two subunits that function as ATPases and another that is a protein kinase, with roles in promoter melting and escape.

1. RNA polymerase does not need a primer- it can initiate transcription de novo 2. The RNA product does not remain base paired to the template DNA strand: the enzyme displaces the growing chain only a few nucleotides behind where each ribonucleotide is added. 3. Transcription is less accurate than replication because it lacks extensive proofreading mechanisms. 4. Transcription selectively copies only certain parts of the genome and makes 1- several thousand copies of any given section. (Replication must copy the entire genome once and only once every cell division.)

Major differences between transcription and DNA replication

torpedo model of termination

Model of termination in which an enzyme that degrades the second RNA as it emerges from the template triggers termination. The free of the second RNA is uncapped and thus can be distinguished from genuine transcripts. This new RNA is recognized by an RNase called RatI in yeast (and Xrn2 in humans) that is loaded into the end of the RNA by another protein (Rtt103) that binds to the CTD end of RNA polymerase. The RatI enzyme is very processive and quickly degrades the RNA in a 5'-3' direction until it catches up to the still transcribing polymerase from which RNA is being spewed. Other factors may be involved in RNA Pol dissociation besides RatI.

The capping of the 5' end of the RNA, splicing, and polyadenylation of the 3' end of the RNA

Once transcribed, eukaryotic RNA has to be processed in various ways before being exported from the nucleus where it can be translated. These processing events include:

regulatory sequences

Other eukaryotic sequence elements behind and typically upstream of the core promoter that are required for accurate and efficient transcription in vivo. These can be grouped into various categories reflecting their location, the organism in question, or their function. All of these DNA elements bind regulatory proteins (activators and repressors) which help or hinder transcription from the core promoter. Some of these sequences can be located many tens or even hundreds of kilobases from the core promoters on which they act.

TAFs (TBP associated factors)

Other subunits of TFIID, some of which recognize other core promoter elements such as Inr, DPE, and DCE. Each TBP is associated with about 10 ——. Two of them bind DNA elements at the promoter, for example, the initiator element Inr and the downstream promoter elements.

Two conserved sequences 6 nucleotides in length separated by a nonspecific stretch of 17-19 nucleotides. The two defined sequences are centered 10bp and 35bp upstream of the site where RNA synthesis starts. The sequences are called the minus 10 and minus 35 elements.

Promoters recognized by RNA polymerase containing sigma factor 70 share the following characteristic structure:

RNA Pol II

RNA polymerase in eukaryotes responsible for transcribing all protein coding genes

RNA Polymerase I

RNA polymerase in eukaryotes that is required for the expression of only one gene, that encoding the rRNA precursor. There are many copies of that gene in each cell, and it is expressed at far higher levels than any other gene, which explains why it has its own dedicated polymerase.

general transcription factors

Several initiation factors that are required for efficient and promoter specific initiation in eukaryotes. In vitro, they are all that is required along with Pol II to initiate transcription on a DNA template without histones. They collectively perform the functions performed by sigma factor in bacteria.

carboxylation-terminal domain (CTD)

The "tail" of the large subunit of Pol II. It contains a series of repeats of the heptapeptide sequence: Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Each repeat contains sites for phosphorylation by specific kinases, including one that is a subunit of TFIIH.

transcription start site

The DNA nucleotide encoding the beginning of the RNA chain and is designated the +1 position. Sequences in the direction in which transcription proceeds are referred to as downstream from the start site and given positive values. (When referring to a position in the upstream sequence, this is given a negative value.)

promoter

The DNA sequence that initially binds the RNA polymerase with any initiation factors required. Once formed, the promotor-polymerase complex undergoes structural changes required for initiation to proceed. The DNA around the point where transcription begins unwinds and only one of the DNA strands acts as a template for transcription. Because RNA polymerase binds promoters in a defined orientation, the same strand is always transcribed from a given promoter.

preinitiation complex

The complete set of general transcription factors and polymerase bound together at the promoter and poised for initiation

TBP (TATA binding protein)

The component of TFIID that binds to the TATA DNA sequence. Upon binding, it extensively distorts the TATA sequence. The resulting —- DNA complex provides a platform to recruit other general transcription factors and polymerase itself to the promoter. Formation of the preinitiation complex containing these components is followed by promoter melting.

polyadenylation

The final RNA processing event which is linked to termination of transcription. Once polymerase has reached the end of a gene, it encounters specific sequences that, after being transcribed into RNA, trigger the transfer of ————- enzymes to that RNA, leading to four events: cleavage of the message, addition of adenine residues to its 3' end, degradation of the RNA remaining associated with RNA polymerase by a 5' to 3' ribonuclease, and subsequently, termination of transcription.

capping

The first RNA processing event which involves the addition of a modified guanine base to the 5' end of the RNA. Specifically, it is a methylated guanine, and it is joined to the RNA transcript by an unusual 5'-5' linkage involving three phosphates. (7-methylguanosine cap/ 7me-G cap). This occurs just after the pre-mRNA emerges from the exit channel of RNA polymerase II. This happens when the transcription cycle has progressed only as far as the transition from the initiation to elongation phases.

initiation

The first step in the transcription cycle during which initial binding of the polymerase to a promoter forms a closed complex. In this form, the DNA remains double stranded and the enzyme is bound to one face of the helix. In the second step, the closed complex undergoes a transition to the open complex in which the DNA strands separate over a distance of 13 base pairs around the start site to form the transcription bubble. The opening of the DNA frees the template strand and the first two ribonucleotides are brought into the active site, aligned on the template strand, and joined together. In the same way, subsequent ribonucleotides are incorporated into the growing RNA chain. Incorporation of the first 10 or so ribonucleotides is an inefficient process, and at this stage (called the initial transcribing process) the enzyme often releases short transcripts of less than 10 nucleotides and then begins synthesis again. Once the polymerase has made a transcript longer than 10 nucleotides, it has said to have escaped the promotor. At this point, it has formed a stable ternary complex containing enzyme, DNA and RNA. This is the transition to the elongation phase.

termination

The step of transcription in which the polymerase stops and releases the RNA product and dissociates from the DNA once it has transcribed the length of the gene. This is caused by sequences called terminators.

isomerization

The transition from the closed complex to the open complex during initiation, which involves structural changes in the polymerase and the opening of the DNA helix to reveal the template and no template strands. This "melting" occurs between positions -11 and +2 with respect to the transcription start site. Two bases in the nontemplate strand of the -10 element (A and T) flip out from their base stacking interactions and instead insert into pockets within the sigma 70 factor protein where they make more favorable interactions without the need for ATP hydrolysis. By stabilizing the single stranded form of the -10 element, these interactions drive the melting of the promotor region. This process is essentially irreversible, and typically guarantees that transcription will initiate.

Initiation, elongation, and termination

Three major phases of the transcription process

activators

Transcriptional regulatory proteins that help recruit polymerase to the promoter, stabilizing its binding there. This recruitment is mediated through interactions between DNA bound activators, chromatin modifying and remodeling factors, and parts of the transcription machinery. One such interaction is with the mediator complex

1. Pincers at the front of the enzyme clamp down tightly on the downstream DNA 2. There is a major shift in the position of the amino terminal region of sigma factor (region 1.1). When not bound to DNA, region 1.1 lies within the active center cleft of the holoenzyme, blocking the path that, in the open complex, is followed by the template DNA strand. In the open complex, region 1.1 shifts and is now outside the enzyme, allowing the DNA access to the cleft. Region 1.1 of sigma factor is highly negatively charged (just like DNA). Thus, in the holoenzyme, region 1.1 acts as a molecular mimic of DNA. The space in the active center cleft, which may be occupied by either region 1.1 or by DNA, is highly positively charged.

Two structural changes that occur in the holoenzyme upon isomerization from the closed to open complex.

TFIIF

Two subunit general transcription factor that associated with Pol II and is recruited to the promotor together with that enzyme (and other factors). Binding of Pol II - TFIIF stabilizes the DNA-TBP-TFIIB complex and is required before TFIIE and TFIIH are recruited to the preinitiation complex.

TFIIE

Two-subunit general transcription factor that has roles in the regulation and recruitment of TFIIH

Torpedo model and Allosteric model

What are the two models of transcription termination?

Rho-dependent and Rho-independent

What are the two types of terminators in bacteria?

This means how many transcripts it initiates in a given time. This is influenced by how well the promoter binds polymerase initially, how efficiently it supports isomerization, and how readily the polymerase can then escape. (Some genes need to be expressed more highly than others and these genes are more likely to have promoters more similar to the consensus).

What does the "strength" of a promoter mean?

Double stranded DNA enters the front of the enzyme between the pincers. At the opening of the catalytic cleft, the strands separate to follow different paths through the enzyme before exiting via their respective channels and reforming a double helix behind the elongating polymerase. Ribonucleotides enter the active site through their defined channel and are added to the growing RNA chain under the guidance of the template DNA strand. Only 8 or 9 nucleotides of the growing RNA chain remain base paired to the DNA template at any given time. The remainder of the RNA chain is peeled off and directed out of the enzyme through the RNA exit channel. The polymerase uses a step mechanism: the enzyme steps forward as a molecular motor, advancing in a single step the distance equivalent to a base pair for every nucleotide it adds to the growing RNA chain. In addition, the size of the bubble (the length of the DNA that is not double helical) remains constant throughout elongation: as 1bp is separated ahead of the processing enzyme, 1bp is formed behind it.

What happens during the elongation phase of transcription?

This impressive feat requires that the DNA template be brought into the polymerase active site and held stably in a helical formation and that the initiating ribonucleotide be brought into the active site and held stably on the template while the next NTP is presented with correct geometry for the chemistry of polymerization to occur. This is particularly difficult because RNA polymerase starts most transcripts with an A, and that ribonucleotide binds the template nucleotide T, with only 2 hydrogen bonds. Thus, the enzyme has to make specific interactions with one or all of the DNA template strand, the initiating ribonucleotide, and the second ribonucleotide- holding one or all rigidly in the correct orientation to allow chemical attack on the incoming NTP. The requirement for such specific interactions probably explains why most transcripts start with the same nucleotide.

Why is it possible that transcription can begin without a primer?