Pre rRNA’ Processing in Prokaryotes:

 

Processing of rRNA, tRNA and mRNA.

 

Processing of rRNA:

Most of the RNAs transcribed are precursor RNAs and they have to be processed to their functional state.  Ribosomal RNAs are synthesized as large precursors and as they are synthesized they are subjected to processing, which involves, methylation (modifications) and pseudouridylation of bases at specific sites by using secondary structural conformations; then cleaving the precursor transcript at specific positions. Methylases, methylate 4-O-guanine bases (GC pairs) and 2-O- group of ribose sugar.  Methylation is very important in the binding of riboproteins to rRNA and also for the folding of rRNA as and when proteins bind to form a ribosomal structure.

 

 

 

Fig_ribo_components

Weaver, Molecular Biology, 2E; http://biology.kenyon.edu/

Eukaryotic Robosomal subunits; https://www.cancerwatch.org

 

Subunits

http://www.icb.ufmg.br/

modulo12_clip_image002_b

http://www.icb.ufmg.br/

 

 

 

Various Kinds of RNA Found in an E. coli Cell

 

Type

Sedimentation
Coefficient

Molecular Weight

Number of
Nucleotides

Percentage of
Total Cell
RNA

mRNA

6–25

25,000–1,000,000

75–1000

~2

tRNA

~4

23,000–30,000

73–94

16

rRNA

5
16
23

35,000
550,000
1,100,000

120
1542
2904

82

Non coding

RNA (NOCORNAc)

Ex. RNA-6sRNA

 

 

60-80

~800

38 intergenic transcripts

 

 

 

 

 

 

 

 

Number of rRNA and tRNA Genes:

 

Species

16s/23s/5s or 18s/28s-5.8s  rRNA genes

5s rRNA genes

tRNA genes

E.coli

7

Embedded in rRNA end segment

~60

S.cerviciae

140

140

250-400

Dictyostelium discoideum

180

180

>850

D.melanogaster:

                         XY:

                         XX:

 

150

250

 

150

150

 

850

850

X.laevis

450-600

24000

1150

Homo sapiens

280

2000

>2000

 

 

 

 

 

Size of Precursor and Processed rRNAs:

 

Species

Precursor

Pre 23s/28s in ntds

16s/18s final in ntds

Percentage

 Of precursor

E.coli

5.6 to 6 kb

2914-3100

1500

80%

Yeast37s

7.2-8.95 kb

3750-3800

1700-2000

80%

Dictyostelium

7.4 kb

4100

1800-2000

 

Drosophila

7-7.4 kb

4100

1800

78%

Xenopus

7.875 kb

4475-4500

1900-1925

79%

Gallus domesticus

11.25 kb

4625

1800

57%

Mus musculus

12.4-13kb

4712-5100

1900-1958

52%

Homo sapiens

13.7kb

5100

1900

 

Plants (general)

7.9kb

3700

1700

71%

 

 

Types

RNA size

Number of ribo proteins

Methylations

70S ribosomal RNAs. (20nm)

 

 Coded by seven RNA operons and 6 protein operons

 

 

30s subunits

16s RNA,

1540-42 ntds

21 (s1 to s21)

Ten,

2’-OH, methyl adenines,

2’-OH, dimethyl guanines,

 help in folding and processing.

50S subunits

23s RNA,

2900 ntds;

5s RNA,

120 ntds

34, L1 to L34

Twenty,2-OH methylations

Total 10 psuedouridines

 

80S ribosomes: 25-35nm

 

Coded by hundreds of genes

 

 

40S subunits

18s RNA;( 1843

Or 1900 ntds)

33;

S1 to s34

43 to 44 methylations at 2’OH groups,

plus conversion of certain Uridine into pseudo-Uridines

60s subunits

28s-RNA;(4718- 4800 ntds);

5.8s RNA;(160ntds);

5s RNA;(120ntds);

Note; 45S rRNA genes are located in nucleolar region of chromosomes 13,14,15,20 and 21

49;

L1 to L45-50

74 methylations at 2’OH of sugars,

Methylation at adenine,

Methylation at guanine, plus conversion of Uridine into pseudo-Uridines.

The number of Uridinylation and methylation sites vary

Mitochondrial ribosomes:

Mammalian-55-56s

 

 

Maize-

Maize-78s,

 

Potato 33s/50s,

30s=12s RNA

50s=16s RNA

12s=954nts,

16s=1558nts

No 5s. in Hu it is imported

 

26s,18.5s,5s

 

26s,18s, 5s

28s-1560 ntds,

48 proteins

-12s -954 nts,29 proteins

 

NO 5s in mammals, but imported

 

Chloroplast ribosomes: 70s

50s and 30s

23sRNA,

16s RNA

5s RNA,

4.5s RNA

 

C. reinhardtii, 70s

50s + 33s ,

 

 

Pea Plastid,70s -

 

50s and 30s

23s

16s

5s

 

 

 

 

 

 

Human- rRNA gene are located on chromosomes 13, 14, 15, 21 and 22 (Nor region), located at 13p12, 14p12, 15p12, 21p12, and 22p12; 5s rRNA genes~ 100 copies, located on chromosome 1-1q42.

Drosophila- rRNA genes located on X -496 genes and Y chromosomes-1445 genes, 5s 100 copies,

Yeast- rRNA genes located on chromosome XII, 150-200 copies.

 

E.coli genome in condensed state and sites of transcription:

 

 

E.coli genome is condensed state with many loops, specific loops containing ceretain gens are engaged in transcriptional activity, transcriptional loci or called transcriptional factories are shown in green. Model linking stable RNA synthesis, RNAP distribution and the dynamic structure of the nucleoid: The E. coli chromosome is represented as blue lines folded in loops, the ori of replication as a black square, the seven rRNA operons as large red circles with letters, and two representative tRNA operons as small red circles. The RNAP molecules are represented as small green circles. For simplicity, only two putative transcription factories/foci, which make the nucleoid more compact by pulling different stable RNA operons into proximity, are indicated here (bottom part of the diagram, large green circles labeled 1 and 2) [adapted from the study by  (Cabrera and Jin, 2003)]. There are seven rRNA  operons and six ribosomal protein operons.

 

 

rRNA operons:

 

In E.coli Ribosomal RNA genes are organized into seven operons, called rrns and ribosomal protein genes into six operons.  Ribose RNA operons, seven of them are dispersed in bacterial chromosomes.  These operons have two promoters, P1 and P2, separated by about 100bps. E.coli rRNA operons are transcribed by two tandem promoters, rrn P1 and rrn P2, with P1 being the predominant promoter at medium to fast growth rates.

 

 

All seven P1 core promoters contain the consensus -10 hexamer (TATAAT) and close matches to the consensus -35 hexamer (TTGACA).  The direction of transcription is same as that replication. The operons are rrn A, rrn B, rrn C, rrn D, rrn E, rrnG, rrn H, in some rrnF is present in place of rrn-H. Transcripts are larger than the final products.  Each precursor RNAs contains non-coding spacer and also tRNA segments are found in spacers.

 

 

http://t2.gstatic.com/images?q=tbn:ANd9GcQU9B2Iy3neChrfSX8XztDPRE-YgjyLKC689MbO6FQs_MLoikP_

Typical rrn operon of E.coli; http://www.jbc.org/

 

http://ars.els-cdn.com/content/image/1-s2.0-S1369527403000389-gr1.jpg

Schematic organization of one of the seven rRNA operon in Escherichia coli: The primary transcript contains all three rRNA species (16S, 23S and 5S), as well as one or more tRNAs transcribed from two promoters, rrn P1 and rrn P2. The transcript is terminated by two terminators, t1 and t2. The rrn P1 promoters consist of three to five binding sites for the transcription activator Fis, an UP element (the binding site for the α subunits of RNAP), and a core promoter element (containing the −10 and −35 hexamers for binding the σ subunit of RNAP).

 

 

E.coli rRNA operons are transcribed by two tandem promoters, rrn P1 and rrn P2, with P1 (in rrnB) being the predominant promoter at medium to fast growth rates.  All RNA’s are transcribed by the same RNAP using sigma 70. All seven P1 core promoters contain exact matches to the consensus -10 hexamer (TATAAT) and close matches to the consensus -35 hexamer (TTGACA).

 

P1 promoter --- (consensus)-- -35 hexamer (TTGACA--(-)10- hexamer (TATAAT)

 

The operons are rrn A, rrn B, rrn C, rrn D, rrn E, rrnG, rrn H, in some rrnF is present in place of rrn-H. Transcripts are larger than the final products.  Each precursor RNAs contains non-coding spacer and also tRNA segments in spacers.

 

http://www.cbs.dtu.dk/dtucourse/27101/images/p1p2.png

Fis = Factor Inversion binding Site. http://www.cbs.dtu.dk/

 

Among the two promoters P1 and P2 of rrnE operon lower concentration of ntds uses P2 otherwise the P1 is used; the start point for P1 is ATP (GTP) and for P2 it is CTP. Fis 98a.a dimer (Factor for inversion and stimulation) binds upstream of rrnP promoters and interacts with RNAP and induces DNA binding. Promoters containing Fis binding site activates rrnB promoters 20-30%; http://www.cbs.dtu.dk/

 

http://ars.els-cdn.com/content/image/1-s2.0-S0923250899001254-fx1.jpg

 

E.coli genetic map with positions of seven rRNA operons

 

E.coli contains 56 tRNA genes. A cluster of nine tRNA genes located in the 1-kb region between ribosomal operons rrnJ and rrnW in Bacillus subtilis has been cloned and sequenced. This cluster contains the genes for tRNA(UACVal), tRNA(UGUThr), tRNA(UUULys), tRNA(UAGLeu). tRNA(GCCGly), tRNA(UAALeu), tRNA(ACGArg), tRNA(UGGPro), and tRNA(UGCAla). The newly discovered tRNA gene cluster combines features of the 3'-end of trnI, a cluster of 6 tRNA genes between ribosomal operons rrnl and rrnH, and of the 5'-end of trnB, a cluster of 21 tRNA genes found immediately 3' to rrnB. Neither the tRNAu'5ZG gene nor its product has been found previously in B. subtilis. In B.subtilis tRNAs are found clustered between rRNA operons. In B.subtilis at the 3’ end of rRNA genes one finds a cluster of 21 tRNAs

 

16s-23s-5s----6tRNAs (trnl)-16s-23s-5s-16s-23s-5s,

16s-23s-5s----9tRNAs (trnJ)-16s-23s-5s-

 

Organization of tRNA and rRNA [Rrn]-Operons in Bacteria:

 

Operon

16s

Spacer tRNA

23s

5s

Spacer tRNA

Mpu site

Rrn-A

+

Ile, ala

+

+

-

86

Rrn-B

+

Glu (2)

+

+

-

89

Rrn-C

+

Glu

+

+

Asp, trp

84

Rrn-D

+

Ile (2), ala (18),

+

+

Thr

70

Rrn-E

+

Glu (2)

+

+

-

90

Rrn-G

+

Glu (2)

+

+

-

56

Rrn-H (F)

+

Ile, ala (18),

+

+

Asp

5

tRNA

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

In E.coli Ribosomal RNA genes are organized into seven operons, called rrns and ribosomal protein genes into six operons. Some tRNA genes are also organized in certain operon clusters.

9s Polycistronic tRNA-such as glyW cysT leuZ, argX hisR leuT proM, and lysT valT lysW valZ lysY lysZ lysQ .

Monocistronic tRNAs- pheU, pheV, asnT, asnU, asnV, and asnW.

 

Some of the tRNAs are organized into polycistronic operons as shown below, eg B.subtilis, otherwise they are located in the middle of 16s and 23s rRNA segments and many at 3’ end beyond 5s segment. A cluster of 21 tRNA genes found immediately 3' to rrnB (fig. shown below.

 

 

B subtilis, rrnB/trnB operon, P and T promoter and terminator respectively;

 

E.coli Operon with its 16s, 23s and 5s coding region often contain tRNA also. These are then processed by specific enzymes.

 

The Maturation of rRNA in E, coli.

 

The primary transcript of the rrnB operon is used here as a schematic model. The other six rRNA-encoding operons are similar in principle but might differ in the number and identity of the tRNAs encoded by the intergenic spacer for example ,rrnB encodes glt; rrnA encodes ileT and alaT), example, rrnB encodes gltT; rrnAencodes ileT and alaT), the number of 5S rRNA-encoding sequences (rrnD encodes two, along with four tRNAs) and the number of Rho-independent terminators.

 

 

Precursor rRNA processing sites; George A. Mackie http://www.nature.com/

 

The precise order of events is not known, but it seems likely that folding and modification of the initial transcript, and the addition of ribosomal proteins occurs before the action of RNase III. Sequences in the leader and spacers anneal to form the processing stems, which are susceptible to the action of RNase III52, 69. RNase III makes staggered cleavages to release pre-16S (17S), pre-23S and pre-5S rRNAs.

 

 

These in turn bind the remaining ribosomal proteins, undergo modifications and are cleaved by combinations of RNase E, RNase G, RNase T and unknown enzymes (at the 3′ end of the 17S rRNA and the 5′ ends of the pre-23S and pre-5S rRNAs). For simplicity, helicases and modifying enzymes are not shown, although they are important for these processes. The maturation of tRNAs involves RNase P and a combination of 3′ exonuclease, including RNase PH, RNase T, RNase D, RNase BN, RNase II and polynucleotide phosphorylase (PNPase). George A. Mackie http://www.nature.com/

 

(1)The segment containing 16S rRNA (small ribosomal subunit) and the one containing 23S rRNA (large ribosomal subunit) are flanked by inverted repeats that form stem structure in the RNA.

 (2)The stems are cleaved by RNase III.  There is no apparent single sequence at which RNase III cleaves ‑ perhaps it recognizes a particular stem structure.  This plus subsequent cleavage events (by an activity called M16) generates the mature 16S and 23S rRNAs.  The rRNAs are also methylated.

 

 

 

RNase III cuts in the stems of stem-loops in RNA; RNA is liberated by RNases P and F. 5S rRNA is liberated by RNases E and M5; http://biowiki.ucdavis.edu/

Excision of rRNAs and tRNAs from 30S precursor RNA; http://biowiki.ucdavis.edu/

 

 

The ends of tRNA in E. coli are produced by the action of three nucleases that cleave the precursor to tRNA. A schematic of the pre-tRNA is shown at the top, with RNA extending from the 5’ and 3’ ends of the RNA that will become the mature tRNA (shown as a cloverleaf). The site of cleavage is indicated by the short vertical arrows above the lines denoting RNA, and they are labeled with the name of the enzyme cutting at that site. The enzymes catalyzing each reaction are listed above or adjacent to the reaction arrows. http://biowiki.ucdavis.edu/

 

Pre tRNA-

 

RNasess P,F,D cut in sequence specific manner. RNase P cleaves at 5’ end of the tRNA, RNase F cleaves at 3’ end and RNase D trim from 3’ direction to the 3’ end of the tRNA

 

 

http://biowiki.ucdavis.edu/

 

The ends of tRNA in E. coli are produced by the action of three nucleases that cleave the precursor to tRNA. A schematic of the pre-tRNA is shown at the top, with RNA extending from the 5’ and 3’ ends of the RNA that will become the mature tRNA (shown as a cloverleaf). The site of cleavage is indicated by the short vertical arrows above the lines denoting RNA, and they are labeled with the name of the enzyme cutting at that site. The enzymes catalyzing each reaction are listed above or adjacent to the reaction arrows.

 

RNaseP- the catalytic RNasep is 375nts long and it is associated with 20kDa protein, Catalytic activity is performed by RNA itself and the protein assists in cleavage reaction. This was one of the first instances discovered of catalytic RNA, and Sidney Altman shared the Nobel Prize for this.

 

Ribosomal RNA and tRNA processing and modifying enzymes:

 

16s Methyl transferase-sun protein-2 copies,

16s rim M,

16S Pseudo Uridine rluA,

16S pseudouridine 516 synthase rsuA,

23S pseudouridine synthase C rluC  2copies,

23S modification pseudouridine synthase D rluD,

Dimethyl adenosine transferase- KsgA,

 

 

S adenocyl methioninet RNAribosyl transferase-isomerase-queA,

tRNA methyl transferse 95-methylaminomethyl-2-thiouridylate) trmU,

tRA guninyl N1-methyl transferase trmD

tRNA guanosine2’omethyl transferasetrnH.

tRNA uacil-5-methyl transferase trnA,

tRNA delta(2)-isopentenyl pyrophosphotransferase miaA,

tRNA pseudouridine synthase truA

tRNA pseudouridine synthase yru B

 

 

Ribosomal Protein Operon (RP): S10 consists of 11 protein coding cistrons

 

 

 

Ribosomal Protein coding operons:

 

L11:  [--] ---L11—L1. [L1 blocks at L11].

Beta: [--]—L10—L7/L12--b--b.[L10 blocks at L10].

Str:   [--]—s12—s7—EF-G—EF-Tu. [S7 blocks at S7].

Alpha: [--]—s1---3—s11—s4---a---L17. [S4 blocks at S13].

S10:  [--]—s10—L3—L4—L23—L2—L19--L22—s3—L16—L29—s17. [L4 blocks at S10]

Spc: [--]—L14—L24—L5—s14—s8—L6—L18—s5—L30—L15. [s8 blocks at S10]

 

http://www.bioinfo.org.cn/book/biochemistry/chapt27/970.jpg

 

 

tRNA genes Embedded in rRNA operons :

 

Operons

tRNA genes encoded in rRNA operon

rrnA

tRNA-Ile, tRNA-Ala

rrnB

tRNA-Glu

rrnC

tRNA-Glu, tRNA-Asp, tRNA-Trp

rrnD

tRNA-Ala, tRNA-Ile, tRNA-Thr

rrnE

tRNA-Glu

rrnG

tRNA-Glu

rrnH

tRNA-Ala, tRNA-Ile, tRNA-Asp

tRNA Operon- Polycistronic

valV valW and leuQ leuP leuV

tRNA Operon-Plycistronic

glyW cysT leuZ and argX hisR leuT proM

tRNA Operon-Plycistronic

2RNAmMet,2RNA1Gln 2tRNA2Gln,tRNAX

 

 

Bacteria synthesize three different rRNAs, called 5S rRNA, 16S rRNA and 23S rRNA, indicating the size of the molecules as measured by sedimentation analysis (see Technical Note 2.2). The three genes for these rRNAs are linked into a single transcription unit (which is usually present in multiple copies, seven for E. coli) and so the pre-rRNA contains copies of all three rRNAs. Cutting events are therefore needed to release the mature rRNAs. These cuts are made by various ribonucleases, at positions specified by double-stranded regions formed by base-pairing between different parts of the pre-rRNA. The cut ends are subsequently trimmed by exonucleases.

Table: Comparison of Ribosomal RNA Modifications: Species from Three Phylogenetic Domains; (E) = Eukaryote, (B) = Bacterium, (A) = Archaeon. Data from [Bachellerie & Cavaille, 1998,Ofengand & Fournier, 1998,Noon et al., 1998].

Species

 

Base Methyls

2'-O-ribose Methyls

Pseudouridines

Total

H. sapiens

(E)

10

107

~ 95

212

X. laevis

(E)

10

99

~ 98

207

S. cerevisiae

(E)

10

55

44

112

E. coli

(B)

22

4

10

36

S. solfataricus

(A)

~ 8

67

9

88

 

Pseudouridine rRNA modifications ($\Psi$) are numerous in eukaryotes and few in bacteria and archaea. Studies of eukaryotic $\Psi$residues show they are found in the most evolutionarily conserved regions of rRNA. $\Psi$are spread throughout SSU rRNA with no clear association with particular functional regions. In contrast, to LSU rRNA $\Psi$residues are clustered in three main regions, all within or structurally associated with the Peptidyl transfer center (PTC) of the ribosome. Individual loss of $\Psi$residues is not lethal [Ni et al., 1997,Gannot et al., 1997], although global loss of pseudouridylation due to mutations in the putative pseudouridine synthase, Cbf5p, causes temperature-sensitive growth impairment. It is thought $\Psi$residues play a variety of roles in the ribosome, some improving translational efficiency, others with undetermined function.

Methylation of RNA occurs at a variety of atoms, nucleotides, sequences and tertiary structures. Strongly related to other posttranscriptional modifications, methylation of different RNA species includes prokaryotic and eukaryotic tRNA, rRNA, mRNA, tmRNA, snRNA, snoRNA, miRNA, and viral RNA. Different catalytic strategies are employed for RNA methylation by a variety of RNA-methyltransferases which fall into four super families. This just outlines the different functions of methyl groups in RNA, including biophysical, biochemical and metabolic stabilization of RNA, quality control, resistance to antibiotics, mRNA reading frame maintenance, deciphering of normal and altered genetic code, selenocysteine incorporation, tRNA aminoacylation, ribotoxins, splicing, intracellular trafficking, immune response, and others. Connections to other fields including gene regulation, DNA repair, stress response, and possibly histone acetylation and exocytosis are pointed out. WIREs RNA 2011 2 611–631 DOI: 10.1002/wrna.79.

Processing means site specific modification at ribose OH ‘sites and  site specific bases by 16s rRNA methylases, this provides resistance to aminoglycosides. N6-methyladenosine, N6-dimethyladenosine, 5-methylcytidine (m5C), 3-methyluridine, and N2-methylguanosine were found.  The 50s 23s rRNA is also methylated at G645 at N1 position. The E. coli 16S and 23S rRNAs contain 11 and 23 modifications respectively, and in many cases the enzymes responsible for these modifications are known. However Pseudouridinylation of rRNA is known, but the mechanism is not clear. Such modifications leads to folding and cutting of rRNA precursors and then bind to respective riboproteins and organize into functional ribosomal subunits.

 

 

RNase-P:

 

 

 

Because of the said features, this P- complex is called Ribozyme. Such Ribozymes are found in eukaryotes and cell organelles; ex. Mitochondria contains ribozyme called MRNP.

 

 

Secondary structure of RNA component of RNase-P;  Structure of the specificity Domain of Bacterial RNase P; Ribbon diagram of the S-domain of B. subtilis RNase P. The P7-P11 stems form a cruciform, with one helix in the cruciform formed by the stacking of P8 and P9, and the other by the stacking of P7, P10 and P11. The GAAA tetraloop at the end of P12 interacts with the tetraloop receptor in P10.1. The highly conserved J12/11-J11/12 module forms a large structure devoid of Watson-Crick base pairs. Andrey S. Krasilnikov et al; https://www-ssrl.slac.stanford.edu

 

Such RNase-Ps are also found in other bacteria, some contain C5 protein with the RNA, archaea contain 4-5 proteins, eukaryotes too contain RNaseP with 9-10 proteins.  Mitochondrial RNase P is just protein no RNA.  Human mitochondrial RNaseP and plastid RNaseP (70kd) contain only protein but no RNA. Mitochondrial ribozyme is called MRNP

 

The Evolution of RNase P: The compositions of characterized RNA-based RNase P enzymes from bacteria, archaea, and eukarya show an increase in protein content with increased complexity of the organism. The sites of interaction between RNase P subunits are not known in most cases and are represented schematically. The structure of the proposed ancestral RNA-only RNase P is not known and is assumed to have the critical structural elements conserved in all forms of RNase P RNA. The composition of the fully characterized mitochondrial RNase P is shown for yeast (S. cerevisiae) and human (H. sapiens). Human mtRNase P is composed only of proteins (mitochondrial RNase P proteins 1, 2, 3) (Holzmann et al., 2008). The third subunit of the human mtRNase P (MRPP3) binds to the two-protein sub complex weakly and may associate dynamically (arrow).

 

Description: Full-size image (47 K)

 

Although key structural elements of the RNA subunit are preserved in various yeast mtRNase P enzymes (solid line), the entire RNA structure is not well defined (dashed line). The Evolution of RNase P(Left). The compositions of characterized RNA-based RNase P enzymes from bacteria, archaea, and eukarya show an increase in protein content with increased complexity of the organism. The sites of interaction between RNase P subunits are not known in most cases and are represented schematically. The structure of the proposed ancestral RNA-only RNase P is not known and is assumed to have the critical structural elements conserved in all forms of RNase P RNA.(Right) The composition of the fully characterized mitochondrial RNase P is shown for yeast (S. cerevisiae) and human (H. sapiens). Human mtRNase P is composed only of proteins (mitochondrial RNase P proteins 1, 2, 3). The third subunit of the human mtRNase P (MRPP3) binds to the two-protein subcomplex weakly and may associate dynamically (arrow). Although key structural elements of the RNA subunit are preserved in various yeast mtRNase P enzymes (solid line), the entire RNA structure is not well defined (dashed line). (Holzmann et al., 2008).

 

 

 

 

Processing of tRNAs found within rRNA Operons:

 

The precursor tRNA, as they are synthesized, they fold into secondary structures, possibly assume a 3-D form.

 

 

 

Post transcriptional Modifications; cleavage of precursor rRNA into rRNA and tRNA; Web text book;

The university of Tokyo;http://csls-text3.c.u-tokyo.ac.jp/

 

Processing of an Escherichia coli pre-tRNA: The example shown above is of tRNAtyr. The tRNA sequence in the primary transcript adopts its base-paired cloverleaf structure and two additional hairpin structures. From 3’ end RNase e/f , the RNase e and F trims from the 3’ end till t reaches 3’A and RNaseP cuts at the 5’ end of the tRNA.

The example shown results in synthesis of tRNAtyr. The tRNA sequence in the primary transcript adopts its base-paired cloverleaf structure (see Figure 11.2) and two additional hairpin structures form, one on either side of the tRNA. Processing begins with the cut by ribonuclease E or F forming a new 3′ end just upstream of one of the hairpins. Ribonuclease D, which is an exonuclease, trims seven nucleotides from this new 3′ end and then pauses while ribonuclease P makes a cut at the start of the cloverleaf, forming the 5′ end of the mature mRNA. Ribonuclease D then removes two more nucleotides, creating the 3′ end of the mature molecule. With this tRNA the 3′-terminal CCA sequence is present in the RNA and is not removed by ribonuclease D. With some other tRNAs this sequence has to be completely or partly added by tRNA nucleotidyltransferase. Abbreviation: RNase, ribonuclease. Based on Turner et al. (1997).

In both bacteria and eukaryotes, tRNA genes occur singly or as multi-gene transcription units.  In bacteria, they are embedded as infiltrators within the rRNA transcription unit. The pre-tRNAs are also processed by a series of ribonucleases, as illustrated in the figure above.  All mature tRNAs must end with the trinucleotide 5′-CCA-3′. Some tRNAs have this sequence already; those that do not, or from which the 5′-CCA-3′ has been removed by the processing ribonucleases, are added by tRNA nucleotidyl transferase.

 

CSLS-Univ. of Tokyo; http://csls-text3.c.u-tokyo.ac.jp/

 

Processing of Bacterial Transcripts with introns:

Consistent with the concept that introns can function as transposable elements, and that nuclear introns derived from self-splicing group II introns, which then evolved in partnership with the spliceosome. These introns are among the select group of catalytic RNAs that have helped spark interest in the role of RNA catalysis in current cellular processes, as well as in the possibility of a primordial ‘RNA world’. The splicing mechanism used by group II introns is similar to that of nuclear spliceosomal introns, which has led to suggestions that the former are the progenitors of the latter. Group II introns have also been linked to retro transposable elements, because some of them have been found to encode a functional reverse transcriptase.

 

It is widely believed that the ribozyme (catalytic RNA) core of group II introns, or some evolutionarily related molecule, gave rise to the RNA components of the spliceosomal splicing machinery of the eukaryotic nucleus. Group II introns seemed to be confined to mitochondrial and chloroplast genomes. The discovery of group II introns both in cyanobacteria (the ancestors of chloroplasts7) and other bacteria I now reported.

 

Group I introns:

Group I introns are widely distributed in protists, bacteria and bacteriophages. Nearly 1500 group I introns found thus far in nature (e.g. in algae and fungi) has only recently been clarified.

Group I introns are common in the 23s rRNA genes of mitochondria and chloroplasts. Often, they encode “homing endonucleases,” which target highly conserved gene sequences and drive inter-organellar intron mobility, even across species and genus lines. Most bacterial 23S rRNA genes show some of these endonuclease-sensitive target sequences. However, only two bacterial 23S rRNA genes are known to contain group I introns. Until such introns are sliced out the rRNAs cannot function.

 

Bacterial mRNAs, unlike eukaryotic mRNA, are polycistronic with spacers in between. The 5’UTR and 3’UTR sequences have role in the stability of mRNAs.  Introns have been found in highly expressed genes in eubacteria, bacteriophages, mitochondria and chloroplasts. The said introns are like eukaryotic Group I and Group II category.  Until they are spliced out mRNAs cannot be translated. Clearly, there must be some means of balancing splicing of bacterial introns with co-transcriptional translation.

 

Semrad and Schroeder (1998) provided the surprising answer that splicing of a group I intron from phage T4 is facilitated by translation of the upstream open reading frame (ORF). This enhancement of splicing is achieved by modulating the long-range conformation of the pre-mRNA. Their results provide useful analogies for the coupling of eukaryotic pre-mRNA splicing with transcription. The introns are found in genes encoding thymidylate synthase (td), ribonucleotide reductase (nrdB) (Belfort 1990), and anaerobic ribonucleotide reductase (sunY, or nrdD) (Young et al. 1994). Self-splicing requires that the intron RNA fold into a unique secondary and tertiary structure (Cech and Herschlag 1996).

 

Description: http://genesdev.cshlp.org/content/12/9/1243/F1.medium.gif

 

Translation enhances splicing of the td group 1 intron from phage T4 in vivo. Recognition of the 3′ splice site (3′ SS) is inhibited by base-pairing of the 5′ exon with the 3′ end of the intron (green); this pairing is prevented by ribosomes bound upstream (Semrad and Schroeder 1998). Ribosomes may also stabilize the folded structure of the intron. Translation of the pre-mRNA terminates at a stop codon (red) after the 5′ splice site (5′ SS). The internal double-strand DNA endonuclease I-Tev I is indicated in blue; the core of the td intron is omitted for clarity. Adapted from Belfort (1990). In bacteria splicing is coordinated with translation.; it is self-splicing. Contain group I and II introns. Translation requires mRNA to be free from sec structure.

 

Splicing of a group I intron from phage T4 is facilitated by translation of the upstream open reading frame (ORF), modulates secondary structure in such a way the splice sites are brought closer so the self-splicing takes place.  Small hairpins can form in 10–100 μsec, and tRNAs can fold within milliseconds.

 

Clostridium difficile: c-di-GMP binding by the Riboswitch induces folding changes at atypical splice site junctions to modulate alternative RNA processing. Some group I introns encode homing endonuclease (HEG), which catalyzes intron mobility. It is proposed that HEGs move the intron from one location to another, from one organism to another and thus account for the wide spreading of the selfish group I introns.

 

Group I and group II introns are not only catalytic RNAs, but also mobile genetic elements. The success of these introns as mobile elements almost certainly relates to their innate self-splicing capability, which enables them to propagate by inserting into host genes while only minimally impairing gene expression. Nevertheless, both types of introns have become dependent on proteins for efficient splicing in vivo to help fold the intron RNA into the catalytically active structure. Base pairing 1-`10 helices-Introns in cyanobacterial mRNAs is a requirement for self-splicing.  Such introns are found in 23s bacterial rRNA- thermophilic bacteria Thermotoga. Group I intron in the recA gene of Bacillus anthracis, require GTP.

 

 

http://nitro.biosci.arizona.edu/

Description: http://2011.igem.org/wiki/images/7/75/PekingR_yxw_f5.jpg

 

Mechanism of group I intron splicing. 5' and 3' exons are in red and blue, respectively. ΩG, terminal intron guanine. G*, exogenous guanosine. (Step I) Nucleophilic attack on the 5' splice site by the 3'-OH of G* in guanosine-binding site (GBS). (Step 2) Nucleophilic attack on the 3' splice site by the free 3'-OH of the 5' exon. (Step 3) Free intron and spliced exons. (Rahul Raghavan and Michael F. Minnick (2009). Group I Introns and Inteins: Disparate Origins but Convergent Parasitic Strategies. American Society for Microbiology 191, 6193-6202) Perking R.

 

 

Group II introns; Group II introns are both catalytic RNAs (ribozymes) and mobile retro elements that were discovered almost ~20 years ago. It has been suggested that eukaryotic mRNA introns might have originated from the group II introns present in the alpha-proteobacterial progenitor of the mitochondria. Bacterial group II introns are of considerable interest not only because of their evolutionary significance, but also because they could potentially be used as tools for genetic manipulation in biotechnology and for gene therapy.

Description: Diagram of structure showing IBS-EBS pairings and bulged "A" utilized during splicing reaction

 

Group II introns are large catalytic RNAs (ribozymes) and mobile retro elements  [reviewed by Pyle & Lambowitz (2006)] .

 

This figure summarizes what is known about the splicing mechanisms and mobility of bacterial group II introns, and describes the recent development of group II intron-based gene-targeting methods. Bacterial group II intron diversity, evolutionary relationships, and behavior in bacteria is very interesting. Group II introns are a class of mobile DNAs consisting of a catalytic RNA (ribozyme) and an intron- encoded protein IEP.

 

 

 

A list of processing enzymes found in cells:

                                               

Name of the Enzyme

Name of the Gene

Substrate

Type of activity

RNase-P

rrPA, B

5’ of tRNA

Endo

RNase-BN

?

3’ of tRNA

Exo

RNase-D

rnD

3’ of tRNA

Exo

RNase-T

?

3’ of tRNA CCA

Exo

RNase-III

PacIp

Rntp Ip

rRNA, snRNAs, snoRNAs and mRNA

Endo

RNase-R

?

rRNA and mRNA

Exo

RNase-E

RnE

5s RNA, tRNA

Endo

RNase-II

Rnb

3’ of unstructured RNA

exo

Polynucleotide phosphorylase

Pnp

Unstructured RNA

Exo,cleaves mRNAs

RNase-H

Rnn-A,B RNA:DNA hybrid

Cleaves only RNA part of cDNA

Endo, sometimes exo also

RNase-F

 

rRNA-tRNA hybrid 3’end

Endo

M16

 

5’ and 3’ end of pre23s rRNA

Endo

M23

 

Pre 23srRNA

Endo

M5

 

5, and 3’ of 5s rRNA

Endo

RNAse MRP

 

5srRNA

 

 

 

 

 

E.coli RNases:

 

 

 

Endonucleases: RNaseE, ,III, P, Z and MazF

 

Act on mRNA

 

RNaseE, C, III

 

Act on rRNAs

 

RNaseP, E Z

 

Act on tRNAs

 

Exonucleases:

 

 

 

PNPase, RNaseH,

R oligoRNase

 

Act on mRNAs

 

RNaseT

 

Act on rRNA

 

 

 

 

 

RNase PH, T,II,O

 

act on tRNAs

 

 

 

 

 

 

 

                                                           

E.coli enzymes;

 

 

 

Detection of 5′- and 3′-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli; Mitsuoki Kawano1et al.

 

Evidence is accumulating that small, noncoding RNAs are important regulatory molecules. Computational and experimental searches have led to the identification of 60 small RNA genes in Escherichia coli. However, most of these studies focused on the intergenic regions and assumed that small RNAs were >50 ntds long. Thus, the previous screens missed small RNAs encoded on the antisense strand of protein-coding genes and small RNAs of <50 nt. To identify additional small RNAs, we (the above authors) carried out a cloning-based screen focused on RNAs of 30–65 nt. In this screen, we identified RNA species corresponding to fragments of rRNAs, tRNAs and known small RNAs. Several of the small RNAs also corresponded to 5′- and 3′-untranslated regions (UTRs) and internal fragments of mRNAs. Four of the 3′-UTR-derived RNAs were highly abundant and two showed expression patterns that differed from the corresponding mRNAs, suggesting independent functions for the 3′-UTR-derived small RNAs. We also detected three previously unidentified RNAs encoded in intergenic regions and RNAs from the long direct repeat and hok/sok elements. In addition, we identified a few small RNAs that are expressed opposite protein-coding genes and could base pair with 5′ or 3′ ends of the mRNAs with perfect complementarity.