10: Transcription: RNA polymerase (2023)

introduction

The released pyrophosphate is cleaved in the cell at 2 Pi, an energetically favorable reaction that drives the reaction toward synthesis. In the presence of excess PPi, the reverse reaction of pyrophosphorolysis can occur. The synthesis always takes place in the 5'-3' direction (relative to the growing RNA strand). The pattern is read in the 3' to 5' direction.

Structure of B. E. coli RNA polymerase

1.This single RNA polymerase synthesizes all types of RNA.

mRNA, rRNA, tRNA

2.It consists of four subunits.

A.nucleus and holoenzyme

a2bb's a2bb' + s

Holoenzyme = a2bb's = núcleo + s = lataStartaccurately transcribe as the appropriate site specified by the sponsor

Nucleus = a2bb' = lataextenda growing strand of RNA

ADistrict Attorneycan be defined in two ways.

1. The DNA sequence required for the precise and specific initiation of transcription.
2. The DNA sequence to which RNA polymerase binds to precisely initiate transcription.

B. Subunits

C.E coliMechanism of RNA polymerase

action modeSfactors

The presence of the s-factor makes the RNA polymerase holoenzyme selective for choosing an initiation site. This is accomplished primarily through effects on the rate of dissociation of RNA polymerase from DNA.

• The nucleus has a strong affinity for common DNA sequences. the t_1/2for the dissociation of the nuclear DNA complex it is about 60 min. This is useful during the stretching phase but not during initiation.
• B. The holoenzyme has reduced affinity for general DNA; decreases by about 10⁴Fold. the t_1/2for the dissociation of the holoenzyme from total DNA it is reduced to about 1 second.
• w. The holoenzyme has a much higher affinity for promoter sequences._1/2since the dissociation of the holoenzyme from the promoter sequences is on the order of hours.

Events at the beginning of the transcript

• The RNA polymerase holoenzyme binds to the promoter to form aclosed complex; At this stage there is no unwinding of the DNA.
• B. The polymerase-promoter complex undergoes the closed-to-open transition, which is a fusion or unwinding of approximately 12 bp.
• w. The primer nucleotides can bind to the enzyme, as indicated by their complementary nucleotides on the DNA template, and the enzyme will catalyze the formation of a phosphodiester bond between them. This polymerase-DNA-RNA complex is called a ternary complex.
• D. Duringfailed initiationthe polymerase catalyses the synthesis of short transcripts of about 6 nucleotides in length and then releases them.
• This phase ends when the nascent RNA of ~6 nucleotides binds to a second RNA-binding site on the enzyme; this second site is distinct from the catalytic center. This binding is associated with the "reset" of the catalytic site so that the enzyme now catalyzes the synthesis of oligonucleotides 7 to 12 in length.
• F. The enzyme is now moved to a new position on the template. During this processsigma leaves the complex. A conformational change in the sigma-associated enzyme leaving the complex allows the "thumb" to wrap around the DNA template, blocking processability.Therefore, the central enzyme catalyzes RNA synthesis during elongation., which continues until "signals" are found indicating termination.

Figure 3.1.8. events at the inauguration

3. Transcription Cycle

4. Nuclear RNA polymerase sites

The . enzyme covers about 60 bp of DNA, with a transcription bubble of about 17 bp being unwound.

d The entry nucleotide (NTP) to be added to the growing RNA strand is attached at the active site for polymerization adjacent to the 3' end of the growing RNA strand as indicated by the template.

The incoming nucleotide binds to the growing RNA strand through a 3'OH nucleophilic attack on the phosphoryl-a of NTP, releasing pyrophosphate.

F. The reaction is progressing (enzyme moving) at about 50 nts per second. This is much slower than the replication rate (about 1000 nts per second).

grams. If the template is topologically constrained, the DNA in front of the RNA polymerase will be coiled (positive supercoiled turns) and the DNA behind the RNA polymerase will be undercoiled (negative supercoiled turns).

The effect of unwinding the DNA template by RNA polymerase is to decrease T by 1 for every 10 bp unwound. So DT = -1 and since DL = 0, then DW = +1 for every 10 sc unwound. This W-increasing effect is exerted on the DNA rather than the polymerase.

The effect of rewinding the DNA template by RNA polymerase is, of course, just the opposite. T increases by 1 per 10 bp rewind. So DT = +1, and since DL = 0, then DW = -1 for every 10bp rewound. This W-lowering effect is exerted on the DNA behind the polymerase, since that is where rewinding takes place.

5. Inhibitors: useful reagents and work references

El. rifamycins, p. Rifampicin: binds to the b subunit to block initiation. The drug prevents the addition of the third or fourth nucleotide, so the initiation process cannot be completed.

How do we know that rifampicin's site of action is the b subunit? Mutations conferring resistance to rifampicin are assigned thisrpoBGen.

B. Streptolidingins: bind to the b subunit to inhibit chain elongation.

These actions of the rifamycins and streptolidingins and the fact that they act on the b subunit indicate that the b subunit is required for the addition of nucleotides to the growing chain.

w. Heparin, a polyanion, binds to the b' subunit to prevent binding to DNA in vitro

(Video) Transcription (DNA to mRNA)

D. Eukaryotic RNA polymerases

1. Eukaryotes have 3 different RNA polymerases in their nucleus.

1. Nuclear RNA polymerase is a large protein made up of about 8 to 14 subunits. MW is about 500,000 each.
2. B. Each polymerase has a different function:

2.subunit structures

The genes and proteins encoding yeast RNA polymerase subunits have been isolated and sequenced, and some functional analyzes have been performed.

B. Some of the subunits are homologous to bacterial RNA polymerases: The two largest subunits are homologous to b and b'. The approximately 40 kDa subunit is the homologue of a.

w. Some subunits are common to all three RNA polymerases.

D. Example of yeast RNA polymerase II:

It is The largest subunit has aDomino Carboxi-Terminal (CTD)with an unusual structure: tandem repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Thr. The yeast enzyme has 26 tandem repeats and the mammalian enzyme has about 50. These can be phosphorylated at Ser and Thr to give a highly charged CTD.

• RNA Pol IIa is not phosphorylated in CTD.
• RNA Pol IIo is phosphorylated at the CTD.

4.RNA polymerases in chloroplasts (plastids) and mitochondria

• The RNA polymerase found in plastids is encoded on the plastid chromosome. In some species, mitochondrial RNA polymerase is encoded by mitochondrial DNA.
• B. These organelle RNA polymerases are much more closely related to bacterial RNA polymerases than to nuclear RNA polymerases. This is a strong argument for the bacterial origin of these organelles and supports the endosymbiont model for the acquisition of these organelles in eukaryotes.
• w. These RNA polymerases catalyze the specific transcription of organelle genes.

E. General transcription factors for eukaryotic RNA polymerase II

It is not known whether the same TAF set is included in the TFIID for all promoters transcribed by RNA polymerase II, or whether some are used only for certain types of promoters. TFIID is the only sequence-specific general transcription factor characterized to date and binds to the minor groove of DNA. It is also used in promoters without TATA, so the role of sequence-specific binding is still under investigation.

3.Summary of common transcription factors for RNA polymerase II.

Factors for RNA polymerase II (human cells)

Roeder, R. G. (1996) TIBS 21: 327-335.

4.TFIIH is a multi-subunit transcription factor that is also involved in DNA repair.

Human factor subunits

Table 3.1.6. RNA polymerase II holoenzyme and mediator

These studies show that RNA polymerase II can exist in many different states or complexes. One is in a very large holocomplex that contains the Mediator. In this state, it initiates transcription exactly when indicated by the TFIID and responds to triggers (Table 3.1.6). The mediator subcomplex appears to be able to dissociate and reassociate with RNA polymerase II and GTFs. In fact, this reassociation could be the step taken to try to identify the mediator. Without a mediator, RNA polymerase II plus GTF can initiate transcription at the correct site (as indicated by TFIID) but is unresponsive to activators. In the absence of GTF, RNA polymerase II can transcribe DNA templates, but it will not start transcription at the right place. Therefore it is responsible for expansion but not for initiation.

Table 3.1.7. Extension of the functions of RNA polymerase II

Figure 3.1.17

If the holoenzyme is the major enzyme involved in the initiation of transcription in eukaryotic cells, then the observed forward assembly pathwayin-vitro(see section d above) may be of minor importancelive. Perhaps the holoenzyme binds to promoters that are targeted simply by binding of TBP (or TFIID) to the TATA box, in contrast to the progressive assembly model, which has a more extensive ordered assembly mechanism. In both models, TBP or TFIID binding is the first step in the complex pre-boot assembly. However, the possibility that the holoenzyme is utilized in some promoters and progressive assembly occurs in others cannot be ruled out at this point.

7.Dianas to activate proteins

The targets of the transcription activating proteins can be some components of the initiation complex. One line of research points to the TAFs in the TFIID as well as the TFIIB as targets for triggers. Thus, activators can facilitate the orderly assembly of the initiation complex by recruiting GTFs. However, the holoenzyme contains the "mediator" or SRB complex that can mediate the response to activators. Thus, activators can serve to recruit the holoenzyme to the promoter. Further studies are needed to determine if one or the other is correct, or if they are separate pathways of activation.

F. General transcription factors for eukaryotic RNA polymerases I and III

1.General transcription factors for RNA polymerase I

The core promoter covers the transcription start site in addition to an upstream control element located approximately 70 bp forward 5'.

B. The UBF1 factor binds to a G+C rich sequence in both the upstream control element and the core promoter.

w. A multi-subunit complex called SL1 binds to the UBF1-DNA complex, again at the core and upstream elements.

D. One of the SL1 subunits is TBP, the TATA-binding protein of TFIID!

RNA polymerase I then binds to this DNA+UBF1+SL1 complex to initiate transcription at the correct nucleotide and extend it to produce pre-rRNA.

2.General transcription factors for RNA Pol III

• The internal control sequences are characteristic of genes transcribed from RNA Pol III (see below).
• B. TFIIIA: binds to the internal control region of genes encoding 5S RNA (internal promoter type 1)
• w. TFIIIC: binds to the internal control regions of genes for 5S RNA (next to TFIIIA) and for tRNAs (internal type 2 promoters)
• D. TFIIIB: TFIIIC binding directs TFIIIB to bind to sequences (-40 to +11) that overlap the transcription start site. A subunit of TFIIIB is TBP, although the TATA box is not required for transcription. TFIIIA and TFIIIC can now be removed without affecting the ability of RNA polymerase III to initiate transcription. So are TFIIIA and TFIIICassembly factors, and TFIIIB is theinitiation factor.

Figure 3.1.18.

RNA polymerase III binds to the TFIIIB+DNA complex to initiate transcription precisely and efficiently.

3Transcription factor used by the 3 RNA polyases: TBP

TBP appears to play a joint role in getting RNA polymerase (I, II, and III) to start in the right place. TBP-containing multi-subunit factors (TFIID, SL1, and TFIIIB) can serve aspositioning factorsfor their respective polymerases.

employees and tasks

• Ross C. Hardison, T. Ming Chu Professor debiochemistry and molecular biology(Pennsylvania State University)

  (Video) DNA Transcription (Advanced)