RNA polymerases:

 

Bacteria and Archaea belong to prokaryotic group, both ha common ancestry and both have similar structural and functional features, but also differ from one another.  Archaea in some structural functional feature is closer to eukaryotes. Bacterial and archaeal RNA polymerases versatile enzymes for they transcribe all types of genes, i.e. tRNA, rRNA and mRNA genes. Though, in terms of promoter elements each of them differs in contents, yet it is the same enzyme with some additional factors responsible for transcription of all the genes.

 

Bacterial RNAPs:

 

RNAP as Holozyme consists of core and accessory subunits having a molecular mass of 476 KD.  In transcription, the eubacterial RNAP holoenzyme, composed of a  sigma subunit and a core enzyme containing the major subunits α2 (RpoA x  2), β (RpoB), and β’ (RpoC), plays a central role. The core enzyme functions in RNA polymerization and requires the σ subunit for specific transcription initiated at the promoter. There are two alpha subunits, one beta, one beta’ and one omega subunit, together they form the core structure.  Another factor called sigma binds and makes it as holozyme and guides the RNAP to its promoter elements.  Different kinds of sigma factors take the enzyme to different but specific gene promoters.  Without sigma factors the bacterial RNAP is an orphan and an aimless wanderer.  Though it can associate with DNA, it gets dissociated. Crystal structure of the Holozyme, up to 25Å resolution, it looks like a puckered palm, with fingers and the thumb on opposite sides can close or open. The groove or the channel found is about 25Å, good enough for holding a ds DNA.  There is also a groove running length wise, which is about 55Å long, but shows branching in the region at which newly formed 5’ end of the RNA threads through it.   The over all dimension is about (100 x 100 x160)Å (or 90x95x150)Å.  The Holozyme exhibits three conformational forms called RP-O (open at higher TM.), RP-C1 (closed at low TM.) and RP-C2 state (closed at 20 0 C).

 

File:Bacterial rna polymerase.png

No higher resolution available. This is a model of bacterial RNAP.
Bacterial_RNA_polymerase.png
(364 × 287 pixels, file size: 9 kilobytes, MIME type: image/png)

 

 

Composition: The enzyme has a complex subunit structure with two configurations designated as core group consisting σ2’ββ & ῲ subunits.  The core group gets associated with sigma factor; this is the functional enzyme called Holozyme. The holoenzyme is involved in the synthesis of most of the cellular RNA.

Bacterial RNAP subunits:

The b’ subunit of bacterial RNAP, which is largest one and plays a role in catalysis, corresponds to the eukaryotic RNAP II subunit RPB1. The bacterial RNAP, b subunit, which is the second largest subunit and also takes part in catalysis and it corresponds to the eukaryotic RNAP II subunit RPB2. The aI and aII subunits of bacterial RNAP, which have identical sequences but different locations, with aI interacting with b and aII interacts with b’, correspond to the RNAP II subunits RPB3 and RPB11, respectively, and play a role in RNAP assembly and transcriptional regulation (Minakhin 2000). Finally, the RNAP subunit ‘w’ corresponds to the RPB6 subunit of RNAP II (Table 1 ).

 

 

Bacterial RNAP

   Eukaryotic RNAP II

b’

RPB1

RPB2

aI 

RPB3

aII 

RPB11

w

RPB6

Table1. Conserved subunits of bacterial RNAP and eukaryotic RNAP II



biochemedit3and5.jpg

Structural similarities between bacterial RNAP and eukaryotic RNAP II:
Structures of Thermus aquaticus core RNAP enzyme at 3.3 A resolution and yeast RNAP II D4/7 at 3.0 A resolution. Adopted from Ebright 2000.

    Bacterial RNAP is approximately 150 A long, 115 A tall, and 100 A wide. The X-ray crystal structure of Thermus aquaticus core RNAP revealed that the enzyme has a shape similar to a crab claw, with the two arms of the claw defining a wide internal cleft between them (Zhang 1999). This cleft is about 27 A in diameters, which can accommodate a double stranded nucleic acid molecule and contains the active center Mg2+. The b’ subunit makes up one arm of the claw and a portion of the base of the cleft, while the b subunit makes up the other arm and a portion of the base of the cleft (Ebright 2000).

  Like bacterial RNAP, Euk-RNAP II has a shape similar to a crab claw, with the two arms of the claw forming a deep cleft between them (Cramer 2000). As Fig. above shows, the relative positions of the conserved subunits in bacterial RNA and eukaryotic RNAP II are the same (Ebright 2000). Hence, RPB1 forms one arm of the claw and a portion of the base of the cleft, while RPB2 forms the other arm of the claw and a portion of the base of the cleft.

Activators and inhibitors: E. coli RNA polymerase has absolute requirements for divalent metal ions such as Mg++, Mn++, or Co++. Rifampicin, streptovaricins, streptolydigin, and sulfydryl reagents are among the inhibitors of E.coli RNA polymerase. Beta-subunit (rpoB) gene mutations were cross-screened against a number of other RNA polymerase inhibitors to correlate susceptibility with specific rpoB genotypes. The rpoB mutants were cross-resistant to streptolydigin and sorangicin A. In contrast, thiolutin, holomycin, corallopyronin A, and ripostatin A retained activity against the rpoB mutants. The second group of inhibitors may be of interest as drug development candidates.

 

 

 

By binding next to RNA polymerase's active center (pink), the potent class of antibiotics called rifamycins (red and yellow) prevents deadly bacterial RNA from elongating. (Credit: Image courtesy of Rockefeller University

 

 

Concentration of RNAPs is approximately 7000 units per cell, and that of sigma factors, which exist as different kinds in their structure and function, is 2300 and odd per cell.  At any given time more than 5000- 6000 enzymes are engaged in transcriptional activity. The rate of synthesis under optimal conditions is about 25 to 45 ntds per second; the error rate is one in 1000,000 ntds incorporation. 

The enzyme perse, not any of its subunits, show explicit 3’—>5’ exonuclease activity, yet they have intrinsic ability to remove mismatched ntds from its 3’ end, by hydrolytic cleavage, which is a proof of its reading function.  They have high processivity and high fidelity. Another significant feature is that in PK, transcription and translation events are coupled.

 

 

 

RNAP components:

 

 

Subunits

Gene

Mol.wt (kd)

Numbers

Function

b

Rpo C

89.5 mpu

155-160 1402 a.a

1

DNA binding

b

Rpo B

89.5 mpu

151-155, 1342 a.a

1

Catalytic

a

72 mpu

36-40 329 a.a

2

Assembly of the core and activation

W (omega)

Rpo Z

82 Mpu

11

1

Regulation and growth of the cell

Sigma

Rpo D

66.5 mpu

70

1

Identity of TATAAT and  --35 region

 

 

 b Subunit: 

The protein, is coded by rpo-B gene, has a M.w. of 150 151 KD.  It has two catalytic sites; one for the first nucleotide incorporation (at 5’end) and the second is for the addition of the next nucleotide for phosphodiester bond formation (called elongation site). 

The b subunit has Zn2^+ binding site.   Rifamycin binds to chain initiation site.  Streptolydigin binds to translocation or elongation site. 

 

In the central region of the protein, a 200 a.a domain, is involved in catalytic activity, but it is also known, that it also participates in transcriptional termination of both Rho dependent and Rho-independent modes. 

 It’s COO^- terminal region is essential for the binding of sigma factor. 

 Its main function seems to be involved in chain initiation and catalysis of polynucleotide formation in template dependent manner.

 

b’ Subunit: 

 

Its Mol.wt is155-160 KD, coded for by rpo-C gene. This is perhaps one of the largest proteins produced by the bacteria.  In general it has more basic amino acids, which mean it is positively charged. 

 It has two Zinc binding sites, and also contains DNA binding stretch and provides a 25 Å groove for it.   The strong affinity for DNA stems from its COO- terminal, which has a zinc finger motif, this region provides strong DNA binding forces.

 

 Beta and b’ together bind to both strands of DNA, but catalytic activity is by b subunit, however it requires b’ subunit for its activity.  The subunits when assembled look like crabs claws. The active center is located at the base of the cleft.  b Subunit makes contact with non-template strand at –8, -9, -36 and –37 and binds to template strand at –32.  Together they also bind to –6 to +1, and from +2 to +11.

 

 

a Subunits: 

 Each RNAP consists of 2a subunits, whose Mol.wt is 34-36 KD each, they are coded for by rpo-A genes.  The C-terminal tail extends to -65 and contacts activator proteins such as CAP/CRP

 

 

 

In 3-D structure it exhibits little longish shape with globular region at their ends.  The proteins have more acidic amino acids. Alpha subunits are required for the assembly of the core complex in a sequence. 

 

These subunits are also required for the assembly of the enzyme as a complex on to the promoter, for these subunits recognize -35 sequences.  Activator proteins, that bind beyond upstream of the –35, interact with CTD domain (carboxyl end) of a units for the activation of the Holozyme.  Mutation or deletion of a subunits fails to activate the RNAP Holozyme.  The N-terminal and C-terminal regions are distinguished by their shape, where the N-terminal region called CDT tail contacts –35 region and the NTD end, i.e. the N-terminal end is complexed with the other subunits of the RNAP.  The two ends ate connected with a hinge like connector.

 

 

 

Sigma factors:

 

These proteins belong to a family of polypeptides and they have very interesting features and play important role in specific gene expression.

 They associate with RNAP subunits and they are also responsible for identifying the correct promoter and positioning the RNAP on DNA in proper context for initiating transcription. Otherwise the RNAP without sig cannot function as an enzyme; it is like a driver without a rudder/conductor. 

 

Sigma factors in E. coli:

 

 

RNAP by itself, can bind to DNA free from any proteins, it moves along the DNA by diffusion, and dissociate without performing transcription.  It can come across different genes and their promoters, but the enzyme fails to recognize which is which, even if it recognizes the promoter it has no bias for a sequence, so it cannot initiate transcription. 

 

 In response to environment cells produce several kinds of sigma factors.  When conditions are favorable, for the growth,  sig-70 is produced in plenty, it is this that drives most of the house keeping genes in active state and also few specialized genes.

 

When conditions change, ex. Nitrogen starvation, or increase in the temperature, changes the situation, where cell survival depends upon its ability to adapt to hostile conditions.  In such situations, signal specific sigma displaces sigma 70 and binds to specific promoters of genes and transcribe.  When conditions revert back to normalcy, sig-70 displaces them. 

 

Sigma is the key factor, that it not only recognize promoter of the gene for transcriptional initiation, but also differentiate the genes to be transcribed.  Different bacterial systems produce their own specific sig factors. In many a situations, different types of sigma factors are produced in cascade manner, to the step wise or sequence wise developmental stages, ex. Sporulation in Bacillus subtilis.

 

Anti-Sigma factors:  Anti sigma factors bind to RNAP and inhibit its activity.  Such factors are found in E.coli, B.subtilis and T4 phages.  Anti-sigma factor 70 such as Rsd in E. coli is present in the stationary phase and blocks the activity of sigma factor 70 which in essence initiate gene transcription. This allows the sigma S factor to associate with RNA polymerase and direct the expression of the stationary genes.

 

Sigma 70:

 

The most common and ubiquitously produced sig-factor in E.coli is sig- 70; and in B. subtilis it is sig-43. 

 There are at least 17 different types of sig-factors, which have been identified and their functions been studied. 

 

 

Different kinds of sigma factors, in spite of structural and functional difference, do show some homology. 

 

The sig-70 is longish extended protein with four domains, called, from the N-terminal, as 1, 2, 3 and 4.  Among the sigma family members domains 2 and 4 are very well conserved and they are 75Å apart.  It positions within the holozyme in such a way it recognizes different elements of promoter region.   The N terminal region is required for associating with beta subunit of RNAP.  The 2nd domain has amino acid sequence, which is aromatic, does not generate any helix turn helix structures, yet it recognizes -10 TATAAT sequence and bind.  The C-terminal 4 th domain recognizes –35 region in sequence specifc manner.  This is also the site at which the N-end of the alpha subunits locates. In this process they intercalate into the major groove of the helix, destabilize it and open-up the helix, which means the DNA melts in that region. 

 

The C- terminal 4th domain has helix turn helix motifs and recognizes -35 sequences and bind.  The – 35 sequences vary from regulated promoters to those of housekeeping gene promoters.     The key components, in recognizing which gene to be identified, and the ability to distinguish one gene from the other, lie in the 4th domain of the sigma factor and the –35 promoter sequence.

 

Figure : The structure of Sigma 70 and its DNA binding site.  (a) Structure of Sigma 70, residues 114 to 448.  PDB ID = 1SIG.  (b) A model for the binding between Sigma 70 and the promoter, based on biochemical studies.  Residues Y425, Y430, W433 and W434 are directly involved in the unwinding (melting) of the double helix.

Sequential assembly of RNAP complex:

a + a-------> 2a,

2a + b -------> 2a 1 b,

2a b + b’ -------> 2ab b’ + ω.

Core enzyme + sig-70 -------> Holozyme.

 

The assembly is sequential and requires all the components and in equal concentrations.  Once transcription is initiated, it is believed the sigma factor dissociates form the RNAP.  But recent studies indicate that sigma factor remain bound but in dissociated form.

 

Members of the sigma70 family of sigma factors have 4 conserved regions (Figure adapted from Fig 7-3, Kornberg and Baker, DNA Replication,

 

Sigma factors of Ec,T4 and Bs and SPo specific and their domains

Region 4: a DNA binding activity that recognizes the -35 promoter motif; this is inferred from allele specific mutations that alter promoter recognition

                   Region 2: Sub region 2.3 and 2.4 form a DNA binding activity that recognizes the -10 promoter motif, region 2 also interacts with core enzyme components; this part of 70 is conserved with the eukaryotic factor RAP30

 

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Positioning of different regions of sigma factor such as 2, 3 and 4, note that the 2nd segment binds to TATAAT box and 4th segment binds to TTGACA

 

 

Chlamydial sigma factor resembles sigma 70, but its functional domains slightly vary

 

While the RNAP covers 60bp or more sigma factor has restricted region to bind to the promoter and RNAP.

 

The RNAP by itself can bind to any promoter but it cannot position itself to a specific promoter and initiate transcription.   Positioning of the enzyme on the promoter exactly in sequence context, in such a way, so as to initiate exactly and absolutely at the same nucleotide, which is predefined and called start site, requires sigma factor.  

  

 

 

Gene

Mol wt

function

Sequence binding

-35

Sequence binding

-10

 

E.coli

 

 

 

 

 

Sig-70

 

 

rpoD

70kd

Most house keeping and few others

TTGACA  <16

18> TATAAT

Sig 54,(rpo N, Ntr A

Glu F)/54 kd

 rpoN/ntrA

54

N2 regulated

-24TTGGNA

 

-12 TTGCA

Sig 32

 

rpoH

 32

Heat shock regulated

CCCTTGAA<13

15> CCCATNTA

Sig 28

fli

 28

Flagillin & chemo taxis

CTAAA   <15>

CCGATAT

Sig 24

rpoE

 24

Extreme Heat shock/oxidative

GAACTT

TCTGA

Sig S

rpoS

38

Stationary phase

 

 

SigFecI, ecI

fecI

19

 irondicitrate transport

 

 

 

B.subtilis

 

 

 

 

 

Sig43 (sig A),rpoD,sigA

sigA

43 

Most genes

TTGACA

TATAAT

Sig 28,(Sig D)

sigD

 28

Flagillin

CTAAA

CCGATAT

Sig 29,(sig E1, spo II GB)

sigE1,SPOii

 29

Sporulation

TTNA

CATTT

Sig H,(sigH, spo H)

sigH/spoH

 

Sporulation

--

--

Sig 32,(sig C )

SigC

 32

Het shock

AAATC

TANTGNTTNTA

Sig 37,(sig B)

sigB

 37

Unknown

AGGNTT

GGNATTGNT

Sig G,(spo 3-G),sigG

Spo3G

 

Fore spore specific

PyGNATpu

CAN (T/A) NTA

Sig F,(spo II AC),sigF

sigF/spoiiAC

 

Sporulation

-35 PyGNATpu

-10 CAN (T/A) NTA

Sig K,(spo IV CB) sigK,

 

sigK/spoivCBIIIC

27kd 

Sporulation mother cell specific

  Not known

  Not known

 

 

Sigma factors, bind to beta subunit of RNAP.  They place the RNAP on to a correct promoter and in correct position in the promoter.   Once the correct promoter has been recognized, it induces conformational changes in the enzyme and activates it.  This leads to the tight binding of the enzyme to DNA and induce the melting of the DNA from –11 to +3 (about 12-14 base pair long) for initiation of transcription. 

 

 

 

 

This figure is another representation of the longish Sigma factor 70

 

 Once the promoter is cleared by the RNAP the sigma factor dissociates from the RNAP, only to be associated with another core enzyme to initiate one more cycle of transcriptional initiation. The sigma 43 of B.subtilis also has similar sequences in promoter regions and similar domains in the sigma factor.   But, in the case of T4 phage sigma factor, it does not possess the C-terminal domain and it does not require –35 sequences for transcriptional initiation, by its T4 RNA polymerase.

 

 

 

 

 

 

 

 

 

 

Different sigma factors in association with RNAP perform different functions in terms of transcription of different genes

 

Omega protein:

 

It is coded for by the gene rpo Z; its Mol.wt is 11 KD. Even during purification of the enzyme, it is difficult to get rid of this omega protein. Absence of this protein from the Holozyme doesn’t affect the function of the enzyme.  But mutations in the gene affect the growth of the cells; growth rate reduces considerably. 

 

This phenotypic effect has been attributed to the disruption of a downstream gene called Spo-T gene. The spo-T gene is involved in the regulation of stringent response to amino acid starvation situations, where the rate of metabolism, rate of RNA synthesis and the rate of translation slow down and the whole cellular events are down regulated.  

 

A substance produced in such situations, called magic molecules, such as Guanosine tetra phosphate (ppGpp), requires omega protein for its activity.  Thus the omega protein has been implicated in transcription.

 

 

Rho protein:

This protein is coded for by rho gene; Mol.wt is 46.  The gene is mapped at 46 mpu position.  It assembles into a hexamer and binds to the 5’ end of newly forming transcripts.  It acts like RNA dependent ATPase and slides along the RNA transcript, and interacts with RNAP at a particular region and terminates transcription.  In Lambda infected bacterial cell one finds such rho-dependent transcriptional termination.  This protein behaves like an RNA dependent helicase.

 

Nus A:

Molecular mass of this protein is 65kd, coded for by nus-A gene. It is involved in termination and anti-termination of transcription.  Very strangely, as soon as the RNAP clears the promoter, sigma is released from the RNAP enzyme (how? and why?) And immediately Nus associates with RNAP.  In fact nus association with RNAP causes decrease in the affinity for NTPS.  But it also facilitates the binding of other proteins, which have anti terminator functions.  When the RNAP with nus is released from the template DNA, sigma factor induces the release of the nus from the RNAP and never vice- versa.

 

RNAPs of Blue green algae: Cyanobacteria are gram negative bacteria (once called Blue Green Algae) and they perform oxygenic photosynthesis. It is the oxygenic photosynthesis has provided oxygen for all respiratory organisms and food for their survival.  Blue green algae are made up of single cells to colonies and filaments, a fascinating group of bacteria, it difficult to call them as bacteria. Fremyella diplosiphon RNAP is made up of five polypeptides (Synechocystis sp.)-RPoC1-161,000Da, RPoB-134,000Da, (91,000Da)?, RPoC1- 72,000 (67Da)Da, and RPoA-41,000 daltons. Anacystid nudulins consists of four subunits- 147kD (b), 125kD (b’),p 86kD and 39kD (alpha).The presence of omega subunit (RpoZ) is not known.

 

There are three groups of sigma factors. Group 1 comprises a principal σ factor that is essential for cell viability. Group 2 (hsp type) is similar to group 1 in molecular structure, but nonessential for cell viability. Group 3 σ factors are an alternative type, structurally different from the group 1 and group 2 σ factors, and are involved in the transcription of regulons for survival under stress. From a structural point of view, σ factors exhibit conserved domains known as regions 1 and/or 2, 3, and 4. Region 1 is subdivided into regions 1.1 and/or 1.2. Region 2 is subdivided into 2.1, 2.2, 2.3, 2,4, and 2.5. Region 2.4 (and 2.5) helps to recognize a 10 (and an extended 10 motif) element. Cyanobacteria Synechocystis contain nine sig factors.

 

 

Archaeal RNA Polymerases:

Three branches of life are shown and their inter relation is sketched.  After life originated in hot springs on this planet archaea came in to existence around 3.9 x10^9 (billion) years ago.  Whether life molecules are still forming in such hot springs found all over the world.  These places are like Urey Millers lab. Archaea and Eubacteria are derived from Last Universal Common Ancestors (LUCA).  Eukaryotes developed from Last Eukaryotic Common Ancestors (LECA). Archaea cells are distinctly prokaryotic. Archaea and eubacteria have many resemblances and many differences.  Interestingly archaea in terms of certain enzymes they resemble eukaryotes. These organisms might be the precursor for eukaryotes.

 

 

 

Three branches of life are shown and their inter relation is sketched.  After life originated in hot springs on this planet archaea came in to existence around 3.9 x10^9 (billion) years ago.  Archaea and Eubacteria are derived from Last Universal Common Ancestors (LUCA).  Eukaryotes developed from Last Eukaryotic Common Ancestors (LECA).

 

Halobacteria sp. strain NRC- WIKI

Methanococcus janaschii,

 

Archaea were first found in extreme environments such as hot springs and volcanic eruptions

 

·         Archaea once thought to be closer to bacteria, but now they are found to have characteristics of both bacteria and eukaryotes.

·         The cell walls of archaea are chemically and structurally diverse and lack peptidoglycan.

·         Archaea cell walls contain a pseudomurein that is different in structure from eubacterial murein, no peptidoglycan.

·         Cells contain gas vesicles and glycogen granules.

·         They don’t contain Nucleus,

·         Archaeal cells contain Histones (like H3+H4) tetramers that can compact DNA into nucleosomes (experimental demonstration) in methanobacterium sp.

·         Archaeal nucleosomes protect ~60bp long DNA; they do contain some histones and they can form nucleosomes?.

·         They don’t have endoplasmic reticulum,

·         They contain 70s ribosomes similar to that of bacteria; their proteins and translation factors are more like eukaryotes.

·         The genomic DNA is circular, remains in supercoiled state.

·         The genome size varies from 0.5Mb to 5.5mb (Methanosarcina barkeri), also contain significant number of plasmids.

·         The genome in Methanococcus jannaschii is 1.7x 10^6bp, there are 1700 protein coding genes.

·         Genes are organized into operons, mRNAs are polycistronic and no introns,

·         rRNA operons with internal transcribed spacer, the number is 1-15.

·         The archaeal RNAPs are now considered closely related to Eukaryotic RNAPII in terms of subunit composition and architecture.

·         The transcriptional regulators activators and repressors encoded by archaeal genome are closely related to bacterial systems or bacterial-like regulatory mechanisms.

·         Basal transcription factors for initiation and elongation are more or less same.

·         RNAPs of this class are large and sophisticated, which interact with DNA/RNA scaffolds.

·         Gene promoter structure in archaea is also more similar to that of eukaryotes than eubacteria in having ‘TATA’ box, although, like the eubacteria, archaea have operons and transcribe these to polycistronic mRNA.

·         Some polycistronic mRNA are cleaved to independent mRNAs and translated.

·         Overall protein synthesis mechanisms of eukaryotes and archaea may be similar.

·         The first amino acid incorporated is methionine not formyl methylated as in prokaryotes.

·         Processing of tRNA and mRNA is same as PK and EK.

·         5’ end of mRNA is processed by RNase P and 3’ by RNaseZ.

·         Introns in tRNA are spliced out and nucleotides are modified.

·         In some cases mRNA contain short poly (A) tail.

·         mRNAs contain 5’ shine Delgarno sequence and binds to 30 ribosome subunit.

·         In some cases translation initiation is like EK.

·         Archaea have a single type of RNAP, responsible for the synthesis of all RNAs. Archaeal RNAP is structurally and mechanistically similar to eukaryotic RNAP II and nuclear RNAP IV and V, and it is especially structurally and mechanistically closely related to eukaryotic nuclear RNAP II.

·         This 12 subunit complex play distinct role in RNAP assembly and catalysis (A’A””, B, D, K, L, f, H, E, G, N and P).  They are homologous to Eukaryotic RNAPII.

·         The enzyme is not inhibited by Rifamycin, but can be inhibited by topoisomerase inhibitors.

·         They don’t produce any endospores; they contain flagella and sex pili.

·         Exhibit transduction and conjugation.

·         Cells possess TCA cycle and electron transport and ATP is synthesized by chemiosmotic process.

·         Ribosomes sensitive to Anisomycin (like EK) and insensitive to Chloramphenicol

 

 

Comparison between Eubacteria, Archaea and Eukaryotes

Eubacteria

Archaea

Eukaryotes

Nucleus

No

No

Yes: membrane-bound

Nucleosomes/histones

No

Yes

Yes

Operons/polycistronic mRNAs

Yes

Yes

No

Introns

No

No

Yes

TATA Box binding protein

No

Yes

Yes

Organelles

No

No

Yes: mitochondria, lysosomes, endoplasmic reticulum etc.

Chromosomes

One Circular

One Circular

More than one

RNA polymerase

One (simple)

More than one (complex)

More than one (complex)

Protein initiator amino acid

N-formyl methionine

Methionine

Methionine

Protein synthesis sensitivity to diphtheria toxin

Insensitive

Sensitive

Sensitive

Peptidoglycan

Yes

No

No

Protein synthesis

  • initiation factors
  • ribosomal proteins
  • elongation factors of Archaea are more similar to those of eukaryotes than eubacteria

 

·         Archaea promoter elements have similarities to that of Eukaryotes.

·         GATACACACAAGATACAGATTATA-----+1>

 

·         Compare to that of bacteria-

---TTGACA---TATATT GACACAACT----+1>

 

·         Transcriptional factors required are TBP, TFB-B binding BRE factors.

·         Transcriptional initiation is similar to that of Eukaryotes.

·         Most of the mRNAs are polycistronic and contain smaller 5’UTR and 3’UTR.

·         Some of the tRNA genes are transcribed as pieces and then they are trans- spliced.

·         Leucine response factors are abundant.  It amounts to ‘reulons’.

 

 

 

Shared with Bacteria

 

 

Shared with

Eukarya

 

Unique to Archaea

No nucleus or membrane-bound organelles

No peptidoglycan

Cell wall structure (for example, some archaeal cell walls contain pseudomurein)

Circular genome

DNA associated with histones[36][37]

Cell membrane containing ether-linked lipids

Genes grouped in operons

Translation initiated with methionine

Flagellin protein structure[38]

No introns or RNA processing

Similar RNA polymerase, promoters, other transcriptional machinery[39][40][41]

Ribosomal structure (characteristics shared with both Bacteria and Eukarya)

Polycistronic mRNA

Similar DNA replication and repair[42]

tRNA sequence and metabolism[39][43]

Cellular size (>100-fold smaller than eukaryotes)

Similar ATPase (Type V)

No fatty acid synthetase enzyme[39]

 

 

 

 

EK

Archa (a)

Bact

classI subunit

Rpb1

A’A’’

B’

 

Rpb2

B

B

 

Rpb3

D

a1

 

Rpb6

K

w

 

Rpb11

L

a2

classII

Rpb4

f

 

 

Rpb5

H

 

 

Rpb

E

 

 

Rpb8

G

 

 

Rpb10

N

 

 

Rpb12

P

 

classIII

Rpb9^1

 

 

 

G down

Rpo13

delta

 

 

 

 

 

 

 

 

 

 

 

 

.

Description: http://www.biologie.uni-regensburg.de/Mikrobio/Thomm/Arbeitsgebiete/relation-1.jpg

Werner F. University College London, Department of Biochemistry and Molecular Biology, Darwin Building, Gower Street, London WC1E 6BT, UK. werner@biochem.ucl.ac.uk.

 

 

 

 

 

                                                                                                Homologous subunits of RNAP from the three domains of life:

                                                                                                The subunit pattern of the RNAP from P. furiosus, pol II of S. cerevisiae and of

                                                                                                the E. coli enzyme are shown. Identical colors indicate homology. Note that

subunit Rpb1 of eukaryotes is split in Archaea into the two polypeptides, A_

                                                                                                and A_. Subunit _ of the E. coli RNAP is homologous to a part of subunit D of

                                                                                                Archaea and of Rpb3 of yeast and to subunits L and Rpb11. The molecular

                                                                                                mass of the subunits of Pyrococcus RNAP is indicated to the left.

Left Archaeal  and Right figure of eukaryotic RNAP II

Schematic representation of interaction of subunits in pol II and the archaeal enzyme: Right panel, modified interaction diagram of pol II subunits based on the crystal structure of pol II according to Ref. 1 and considering the interactions of Rpb3 and Rpb7 according to Ref. 2. The color code is the same as in Figs. 1 and 4. Left panel, interaction diagram of Pyrococcus subunits based on Far-Western analyses. The homology of subunits to pol II is indicated by the color code. The thickness of the lines connecting the subunits gives an estimation of the strength of interactions. Connecting lines in blue color indicate an interaction established by one labeled probe, connecting lines in red color indicate interactions established by both interacting partners as probe. Crit Rev Biochem Mol Biol. Author manuscript; available in PMC 2012 February 1.

 

Comparison of Bacterial RNA polymerase and Eukaryotic RNA Polymerase II


    RNA polymerase is an enzyme that is conserved in all living organisms. Because bacterial, archaeal, and eukaryotic RNAPs are large enzymes comprising many subunits; they are part of a conserved protein family called “multisubunit RNAP family (Ebright 2000).” The bacterial RNAP core enzyme is made up of five subunits (b’, b, aI, aII, and w), with a molecular mass of approximately 0.35 MDa (Minakhin 2001). Eukaryotic RNAP II consists of these five conserved subunits and seven additional subunits, with a molecular mass of about 0.5 MDa. It has been shown that the subunits of the bacterial RNAP core enzyme and the subunits of eukaryotic RNAP II exhibit sequence, structural, and functional homology (Minakhin 2001). Bacterial w subunit of RNAP is a homolog of Eukaryotic RPB6.

Eukaryotes have multiple types of nuclear RNAPs, each responsible for synthesis of a distinct subset of RNAs. All are structurally and mechanistically related to each other and to bacterial RNAP (wiki).

Chloroplasts in eukaryotic cells contain two types of RNAPs; one structurally and mechanistically similar to bacterial RNAP ("plastid-encoded polymerase with four subunits PEP "). Chloroplasts also contain a second type, structurally and mechanistically unrelated, RNAP ("nucleus-encoded polymerase"); member of the "single-subunit RNAP" protein family coded by nucleus (NEP).  Zea mays chloroplasts in green tissues contain nuclear coded sig1 and sig2 (50kDa in green and -60kDa in both etioplasts and green plastids) proteins. Sig 5 and sig6 are also found in Oryza sativa.

Mitochondrial RNAPs: Eukaryotic cells contain specialized symbiont organelle called mitochondria.  This has its genome with just 13 protein coding genes and 22tRNA genes plus two rRNA genes. During endosymbiontic process oxygen respiring bacteria has lost more than 99% of it genome to the eukaryotic cell, yet it has retain very crucial genes without which the parent cell is hapless and helpless. Mitochondria contain an nuclear coded single subunit RNAP which is structurally and mechanistically unrelated to other RNAPs, member of the "single-subunit RNAP" protein family, similar to that of T7, Sp6  and T3 phage RNAPs.

RNA polymerases in Viruses:

Orthomyxovirus- It is structurally related to bacterial, archaea and eukaryotic RNAP I-IV. Some viruses use single subunit DNA dependent RNAP and related to eukaryotic mitochondria and chloroplasts.  There are single strand transcriptases or RNAPs. There are RNA dependent RNAPs, which convert (-) strand to positive strand and vice versa. There are DNA dependent Viral RNA polymerases.

Viral coded- RNA polymerases:

Picorna virus:   The gene 3D codes for nonstructural protein that acts as viral RNAP.  The 3D codes for RNA dependent RNA polymerase.

T7 Phage:  codes for a RNAP of  99kDa and it uses promoter sequences, even Sp6, T3 RNAPs have the same structural and functional similarities.

--I -------(-35) TTGACA----------(-10) TATAAT------+1>>>

 

Herpes simplex:  The virus codes for an RNAP.  It uses three different promoters such immediate early, early and late promoters.

 

Description: http://ars.els-cdn.com/content/image/1-s2.0-S0378111901005121-gr2.jpg

 

Adeno virus:

There are different promoters such as early1A and 1B and late they are transcribed with time. The transcription is performed by ADV RNAP IIIc protein

Description: Full-size image (47 K)

Figure 1. Schematic illustration of the HAdv5 genome, which consists of a 36 kb linear double-stranded DNA that is covalently linked to viral terminal proteins (TP) at the 3′ ends. The viral genome contains five early transcription units (E1A, E1B, E3, E4 and E2) and one major late transcription unit. The major late transcript is differentially processed into five groups of late mRNAs (L1–L5), to which three leader sequences are attached by RNA splicing. The late mRNAs code for various viral structural proteins. The left-hand 14% of the genome contains the E1A and E1B genes, which constitute the transforming genes. l-E1A indicates the large E1A protein and s-E1A indicates the small E1A protein. The early region E4 of different HAdv species encodes multiple proteins, of which at least three, Orf 1, Orf 3 and Orf 6 (indicated by 1, 3 and 6), exhibit transforming activities.

 

Retro Viral coded RNA dependent DNAP:

 

There are two RNA genomes and they code for Reverse transcriptase, that use RNA to generate cDNA.  The enzyme uses promoter elements typical of eukaryotes.

 

Promoter elements  in LTR region of the retroviral genome.

 

5’ –AP1-NFAT—NRF---NFkB-Sp1--CCAATTATA---TATA----R

 

Given that DNA and RNA polymerases both carry out template-dependent nucleotide polymerization, it might be expected that the two types of enzymes would be structurally related. However, x-ray crystallographic studies of both types of enzymes reveal that, other than containing a critical Mg2+ ion at the catalytic site, they are virtually unrelated to each other; indeed template-dependent nucleotide polymerizing enzymes seem to have arisen independently twice during the early evolution of cells. One lineage led to the modern DNA Polymerases and reverse transcriptase (RTs), as well as to a few single-subunit RNA polymerases from viruses. The other lineage formed all of the modern cellular RNA polymerases.  However there Polymerases that adds CCA to tRNA 3’ends.  Some polymerases add Poly-A to the 3’ends of mRNAs.: M. F. Symmons, G. H. Jones & B. F. Luisi

     In 1955, Marianne Grunberg-Manago and Severo Ochoa reported the isolation of an enzyme that catalyzed the synthesis of RNA. Their work built upon the earlier work of Jerard Hurwitz & J.J. Furth who performed experiments to see if isolated E. coli protein fractions could polymerize radioactively labeled nucleotides. Later Grunberg-Manago & Ochoa tested a protein fraction that could make RNA. For this work, Ochoa shared the 1959 Nobel Prize in Medicine with Arthur Kornberg (who received the Prize for his work on DNA polymerase- I). This enzyme works as polymerase without template.

This enzyme could convert ribonucleotide diphosphate into RNA:

(RNA)n + NDP --> (RNA)n+1 + Pi

However, the enzyme had a number of unsettling properties. It does not need a template; and, it could use as little as 1 NDP or as many as 4 NDPs as substrate. In fact, the sequence of the product RNA depended entirely on the number and concentration of substrate NDPs.

These are not the properties of an enzyme that must faithfully copy the genetic material for expression!

We now know that Grunberg-Manago and Ochoa had isolated the enzyme polynucleotide phosphorylase which usually catalyzes the breakdown of RNA - not its synthesis! i.e., it is a ribonuclease; it can polymerize nucleotides in vitro.

Reference: Structure, 8:1215-1226, 2000

 

Description: PNPase is a disk-like trimeric exo ribonuclease. This side view of the trimer shows the interface between two subunits. The cores of subunits are constructed from two homologous domains shown in shades of red and blue. The lower and upper accessory domains are in green and grey respectively.

 

 

This view of the channel through the centre of the trimer channel shows side chains of the conserved 'FFRR loops' that may act as a mechanism to entrap RNA substrate and select against base-paired segments. This provides a possible structural explanation for both the high processivity and the regulation of the PNPase activity by RNA secondary structure. 

 

 

Prokaryotic DNA polymerases;

Bacteria have 5 known DNA polymerases:

 

 

Eukaryotic DNA polymerases;

Eukaryotes have at least 15 DNA Polymerases:

None of the eukaryotic polymerases can remove primers (5'->3' exonuclease activity), that function is carried out by other enzymes. Only the polymerases that deal with the elongation (γ, δ and ε) have proofreading ability (3'->5' exonuclease).