3.3.6. Transcription Elongation
In elongation process RNA pol II polymerize the nucleotides complementary to template and form RNA chain. RNA polymerase does not transcribe continuously inspite of pause some specific site and backtrack some nucleotide, if pause for short time than polymerase easily again start transcribing, if pause long than require elongation factor for restart the transcription.
Two factors negative elongation factor (NELF) and DSIF (DRB sensitivity inducing factor contain two subunit SPt4 and hSPT5) are responsible for stabilizing the pause RNA polymerase, DSIF bind to phosphorylated serine present on tail of polymerase and recruit NELF, which placed in front of RNA polymerase (downstream) and capping enzyme, which capped the 5’ end of mRNA, so capping co-transcription process.
After completion of capping process, NELF removed by a positive transcription elongation factor-b (PTEF-b), which work as kinase and phosphorylate 2nd serine of heptapeptide repeats. Phosphorylation of 2nd serine decrease the affinity of NELF hence they dissociate and DSIf remain there. DSIF subunit hSPT5 upon phosphorylation stimulate the transcription. PTEF-b also recruit TAT-SF1, which in turn recruit splicing machinery.
Transcription factor IIS, another accessory factor comes in elongation that limiting the time length of polymerase pauses by stimulating the overall rate of transcription. Transcription factor IIS also having role in proofreading, when some wrong nucleotide get attached, pause in transcription also occur. At that time transcription factor IIS stimulate the internal RNAS activity of RNA polymerase and work along with it within its active site through the help of two acidic amino acid which recruit two mg+2 ion involved in catalysis. RNA polymerase trims some nucleotide including wrong nucleotide from 3’OH and create new 3’OH require to restart the transcription. Transcription factor IIS show homology to Gre factor involves in hydrolytic editing in prokaryote transcription.
3.3.7. Transcription Termination
In eukaryotic transcription termination process a termination sequence (AAUAAA and UGUGGU) are present on emerging RNA. This AAUAAA sequence is recognized by CPSF (cleavage and polyadenylation specific factor-which is a tetramer) and another protein CstF (cleavage stimulating factor) bind at UGUGGU sequence, a dinucleotid AC is present between these both sequence. Two cleavage protein cleavage protein-I and cleavage protein-II now bind on C and A respectively. After binding of CPSF and CstF they interact with each other. Now cleavage protein I and cleavage protein II cleave the phosphodiester bond present between C and A thus newly synthesized m-RNA molecule is free.
After sometime RNA pol II is dissociate from template and remaining RNA is degrade.
In this process, 250-300 nucleotide long adenine sequence added on newly synthesized RNA molecule. Two enzyme PAP I slow polymerase add 10-12 adenine and PAP-11, is a fast poly (A) polymerase and add 100 - 200 adenine on 3' OH of newly synthesized m-RNA and those adenine protected by other factor PABP (poly Adenylate binding protein). This adenylation is called as tailing.
Polyadenylation help in nuclear export of m-RNA polyadenylation provide stability to m-RNA. If polyadenylation not occur m-RNA is degraded by RNase. Poly A tail also provide binding site for translation factor and serve as recognition signal for initiation of translation.
3.5. Capping of m-RNA
Capping is co transcriptional modification during transcription when 5' and is emerged a short pause occur in transcription, this pausing of transcription essential for capping process. Firstly g-phosphate of 5' adenine is removing by g-phosphatase enzyme than guanylyl transferase enzyme transfer GMP from GTP to 5' diphosphate of mRNA and form guanosine 5' – 5' triphosphate structure. So endonuclease cannot able to cut this 5' – 5' linkage because if is not a phosphodiester bond (endonuclease cut only phosphodiester bond). An another enzyme guanine-7 methyl transferase transfer methyl group from s-adenosyl methionine to N7 position of guanine at 5' end of RNA. This capping of 5' end is called as CaP O (cap zero). This is the first methylation step occur in all eukaryotes.
Cap-1 : Methyl group add on 2' OH of 2nd nucleotide at 5' end.
Cap-2 : Methyl group add on 2' OH if 3rd nucleotide at 5' end.
Cap 0 normal feature of mRNA but additional capping like cap1 and cap-2 is responsible to increase the life span of mRNA, as much as cap mRNA as much as life span of mRNA increase. Capping also help in nuclear mRNA transport, translation (provide binding site for ribosome) and provide stability to mRNA.
3.6. Transport of m-RNA in cytoplasm
Some protein factors are require for transportation of mRNA (e.g., UAP-1, UAP-II, LOSS, Mtr, exportin). Mtr-1 bind at 7 guanine, loss bind at poly A tail and UAP bind at junctions of exons, Exportin also bind to mRNA, so capping and tailing is necessary for transport of mRNA. If cap is removed from m-RNA, m-RNA remains in nucleus.
If mutation occurs in LOSS, exportion UAP-I, II, III and Mtr-1, the rate of transport decrease but transport not stop. Means some other factor involved in transport of mRNA which transport only spliced m-RNA, hn-RNA containing introns are retaining in nucleus. These factors called retention factors.
3.7. Promoter of RNA pol I
RNA pol I promoter contain a core promoter spanning between +20 and –45 from start site which is rich in GC content but ‘Inr’ sequence of core promoter is rich in AT content. An upstream control element (UCE) or upstream promoter element (UPE) also present between –100 to –180 from start site. UCE equally divide within two site, first, site A to which SL1 (containing 3 TAFs and TBP) initiation factor bind and second, site B to which UBF another initiation factor bind. In presence of UBF, SL1 binds to site A and then recruitment of RNA polymerase occur by UBF after that transcription start from core promoter.
Genes which posses promoter I transcribed into r-RNA except 5s rRNA.
3.8. Promoter of RNA pol III
There are 3 different types of promoter III for transcription of t-RNA, 5s RNA and other type of RNA.
Type-I promoter III: Genes which have type I promoter III transcribe into 5s rRNA. In type I promoter Box A and Box C present in coding region. Box A located between +50 to +70 from transcription start site. Box c present between +80 and +90 region. There are 3 transcription factor TF III A, TF III B and TF III C required for 5s rRNA transcription.
TF III A is bind on promoter first and followed by factor C and factor B. than pol III recruit by TF III B.
Type II promoter III : Type II promoter III present in t-RNA gene. In type II of promoter III box A and box B found in downstream of start site, box A lie between + 10 and + 20 and box B located between +50 and +60. Box A sequence region form D loop of t-RNA and Box B form TCG loop of t-RNA. In synthesis of tRNA two transcription factors TF III B and TF III C are required TF III C bind first than TF III B is bind on promoter. Now RNA Polymerase III binds on promoter. TF III B cannot able to bind on DNA. It binds on TF III C and thus a pol III transcribed t-RNA gene in multiple round (up to so round).
Termination of tRNA is occurred as Rho independent manner.
Type III promoter III: This type of promoter present in U6 snRNA and contain oct sequence, PSE sequence and TATA sequence. TATA box present at –30 from start site. PSE located at –60 positions.
3.9. Synthesis and Processing of r-RNA in eukaryotes
Eukaryotes have 4 type of rRNA, 28 srRNA, 18 SrRNA, 5.8 srRNA and 5 srRNA. Among these ribosomal RNAs three r-RNA (28S rRNA, 18S rRNA and 5.8S rRNA) transcribed from a single nucleolar gene. As noted earlier 5S rRNA gene consists promote III and transcribed by RNA Pol III.
28S rRNA, 18S rRNA and 5.8 S rRNA gene consist promoter I and RNA Pol I transcribed this gene as a pre rRNA [45S] of 13.7 kb. RNA Pol I require two other factors, factor B and factors (ask). After bind of these TC factor RNA Pol I bind on promoter and form initiation complex. Thus r-RNA gene transcribed into 45S pre-rRNA.
Ribosomal protein assembled on this 45S pre r-RNA during transcription. This whole process occurs in nucleolus (nucleolus is the compartment where rRNAs are main ribosomal protein assembled). In pre rRNA some spacer sequence are present between 28S rRNA, 18S rRNA and 5.8S rRNA. These spacer are remove from pre rRNA and some chemical modification also occur in pre-rRNA, this is known as rRNA processing.
Thus rRNA processing involves:
(i) Chemical modification of done with the help of different type snoRNAs (small nucleolar RNAs), which base pair with rRNA and this base pair region serve as a site for modification like methylation upto 100 nucleotide undergo methylation.
(ii) Cleavage and trimming process done with the help of endonuclease, which responsible for cleavage of large pre-rRNA and exonuclease to make mature rRNA by trim the cleavage product.
In first step, 45S rRNA spacer sequence in removed. After removing spacer sequence 45S rRNA converts in 41S rRNA. In second step 41S rRNA cleaved into 2 fragments, one is 20s and second is 32S. 20S fragment contain 18s rRNA and 32s contain 28s and 5.8s rRNA. Now 20s rRNA gives 18s rRNA by trimming process, And 32 intermediate give 28s rRNA and 5.8 rRNA.
After trimming 5.8S rRNA paired with 28s rRNA within 5 minute of processing 18s transport into cytoplasm and assembled as 40s subunit of ribosome. And within 30 minute 28S rRNA, 5.8S rRNA hybrid and 5S rRNA which transcribed from different transcription unit also assembled with this hybrid. Now this complex also transport to cytoplasm and where it assembled as 60S subunit.
Splicing of rRNA (28s, 18s, 5.8s rRNA) is auto splicing or self splicing. In 5s rRNA splicing process not takes place.
3.10. Processing of Eukaryotic tRNA
Nuclear pre tRNA of eukaryote contain intron recognize by common secondary structure in tRNA and remove by following mechanism, firstly intron boundary get recognize and then removal of intron by cleavage of phosphodiester bond on both splice site carried out by endonuclease ( SEN54, SEN2, SEN34, SEN15).
As result two tRNA half-molecule comprising 5'-OH ends and 2',3'-cyclic phosphate (2',3'- P) produce which stay together via H-bonding. Linear intron having 5'-OH and 3'-phosphate (3'-P) ends also produce.
In next step, a cyclic phosphodiesterase (CPDase) open 2',3'cyclic PO4 and produce 2'-PO4 group and a 3'OH group. after that a terminal polynucleotide kinase enzyme phosphorylates the 5'-OH of the 3'-exon using the - PO4 of GTP, this step require ATP, after that formation of the 5'-3'-phosphodiester bond proceeds with the help of tRNA ligase and ATP. In last 2’ phosphate removes by 2’ phosphatase. Nuclear pre tRNA Intron comprising complementary structure with anticodon of the tRNA. Eukarya and Archaea endonucleases are closely phylogenetically related.
Processing of Prokaryotic rRNA: In prokaryotes, 30 S pre rRNA form, which undergo cleavage, trimming and chemical modification like eukaryotes. Prior to cleavage 30 S precursor metylated at specific base than cleavage produce 17S rRNA and 25S rRNA intermediate. Cleavage reaction also called primary processing carried out by RNase P, RNase E/F and RNase III, after that secondary processing also called trimming takes place as a result 16 S and 23 S produce by specific nuclease reaction carried out by RNase M. 5S rRNA produce by 3’ splice site and from the mid section of 30 S precursor one or more tRNA also produce.
Processing of Prokaryotic tRNA:, tRNA introns in eubacteria are group I and Group II self-splicing introns, with splicing cleavage, trimming and chemical modification comes in prossessing of pre-tRNA precursor which comprising extra sequence. 3’ end create by endonucleolytic action of RNase E/F after that trimming upto seven nucleotide done by exonuclease RNaseD. In last tRNA nucleotidyltransferase add CCA at newly form 3’ terminus, CCA not encode by genome. 5’ end created by the action of a RNaseP (ribonuclease P).
3.11. Non coding RNA
It is an RNA molecule that cannot be translated into protein. They regulate the gene expression at the level of transcription, RNA processing and translation. It includes tRNA, rRNA, snoRNA, miRNA, snRNA, siRNA, scaRNA (small cajal) ,piRNA (piwi interacting RNA), exRNA (exosomal RNA). They perform many vital functions and regulate the gene expression.
Ribosomal RNA are the cellular machinery which is used to translate the mRNA into proteins. They are also called as ribonucleoproteins (RNPs) assembled in nucleolus.
In prokaryotes the ribosome size is 70S in which 50S being the larger subunit and 30S smaller subunit. Smaller subunit contains 16s, 3’of which binds with the shine dalgarno sequence of 5’mRNA. In eukaryotes, the ribosome size is 80S in which 60S being the larger subunit and 40S smaller subunit. The 28 s,S.8S, 18S rRNA are transcribed by a single transcript(45S) separated by 2 internally transcribed spacers.
They are transcribed by the RNA pol I except the 5S rRNA which is transcribed by RNA pol III. rRNA play an important role in evolution and are sequenced to identify the taxonomic group.
They are adapter molecule that makes the codon in mRNA strand to the corresponding aminoacid. It is also known as soluble RNA. There are 61 different typer of tRNA for 61 sense codons.
The secondary structure of tRNA is like the cloverleaf and the tertiary structure is like L shaped.
It consists of following:
- 5’ terminal phosphate group.
- D loop containing modified base dihydrouridine.
- Anticodon loop containing the anticodon.
- TΨC loop contains pseudouridine.
- Acceptor arm contains CCA 3’ terminal group where the aminoacid is loaded by the aminoacyl tRNA synthetase. An aminoacyl tRNA synthetase(aaRS) catalyses the attachment of cognate AA( perfectly matched) onto its tRNA.
Loading of cognate AA will be perfectly binded and they have least rate of dissociation and NON cognate(Imperfectlly matched) has high rate of dissociation. So by means of kinetics of codon-anticodon pairing, ribosomesareable to distinguish the non cognate tRNA from cognate one. This is called as kinetic proof reading.
Theses RNAs interact with the piwi family of proteins. They play an important role in transposon silencing in germline and somatic cells.
In prokaryote, when sufficient has been synthesized, endonuclease degrades the mRna and ribosome get stalled on the mRNA.
Therefore to release the stalled ribosome tmRNA come into action.
It has following 2 features
- Like mRNA it carries stop codon
- Like tRNA it carries amino acid
The resulting polypeptide will be degraded by UPS and ribosome will be released and recycled.
snoRNA (small nucleolar RNA) :
They guide the chemical modification of rRNA, tRNA and snRNA by2 different members of family. One is C/D Box which modifies by methylation (addition of methyl group) and the other is H/ACA box which do so by pseudouridylation, addition of isomer of nucleoside uridine.
snRNA (small nuclear RNA) : their average length is 150 nucleotide. Their primary function is the splicing or the processing of pre-mRNA in the nucleus.
They also aid in regulation of transcription factors or RNA polymerase II as well as maintain the telomeres.
It can be associated with a set of proteins and form complexes called as snRNPs.
miRNA (micro RNA) and siRNA (small interfering RNA) :
They function in transcription and post transcriptional regulation gene expression and they do so by base pairing with complementary sequences within the mRNA and results in gene silencing, processes called as RNA interference.
3.12. RNA interference :
All cells carry the exact same genome still we end up with so many variations. This is because the transcription of many eukaryotic genes are silenced or repressed. Some genes which are transcribed into mRNA are never get translated. It is a technique to control the gene expression. It involves 2 important RNA: miRNA and siRNA. They select the target mRNA and chop them up so there is no protein produced. If there is any production of dsRNA inside the cell it can produce siRNA, miRNA andshRNA. Theses RNA lead to the silencing of the gene i.e. called as RNA mediated gene silencing with the help of protein complex called as RISC( RNA induced silencing complex).
It is a hairpin loop structure formed by the base pairing within mRNA of 100-200nts. An enzyme Drosha and DGCR8 combine together abd act on pri-miRNA and cut some part of pri-miRNA. It is termed as pre-miRNA now. Drosha is Nuclear Rnase specific for dsRNA and DGCR-8 is a dsRNA binding protein. A protein known as Export in 5 transports this pre-mi RNA out of the nucleus to the cytoplasm. In cytoplasm, Dicer (RNase III/ds RNase) and TRBP bind with it and cleave the hairpin loop and make it linear and generates the3’ overhangs and 5’ mono phosphate. Now this is called as miRNA: miRNA* duplex off 22 nts. The passenger strand of this miRNA binds with the target mRNA. Argonauta protein, one of the proteins in RISC activated and cleaves the mRNA.
In animal cell, miRNA is imperfectly paired so it causes the deadenylaation of thepoly A tail. This causes the mRNA to be degraded soon and reduce the translational ability. In plants, miRNA is perfectly paired with mRNA and degrades it.
siRNA are small double stranded RNA molecules( about 20 base pairs in length) generated by cleavage of ds RNA by an enzyme called Dicer. Source of this dsRNA can be Exogenous or endogenous. Exogenous source can be injected dsRNA from outside. Endogenous source is transcription of both the sense and antisense strands of DNA from the same loci so they have the complementary base pairs.
dsRNA is transported out of the nucleus to cytosol. Here the Dicer enzyme produces 3’ overhangs and 5’ monophosphate. This is called as the processed siRNA. Slicer and argonaute (RNaseH) protein of RISC binds with siRNA and unwinds it and degrades the passenger stand and remain binded with the outer strand called as guide stand. Slicer present in C. Elegans. Then this complex binds with the target mRNA and degrades it.
3.13. DNA-binding domain
A DNA-binding domain is a protein structure that has a high affinity for DNA. It is an independently folded protein domain. A DBD can recognize a specific DNA sequence (a recognition sequence).DNA recognition by the DBD can occur at the major or minor groove of DNA, or at the sugar-phosphate DNA backbone.
Types of DNA-binding domains
1. Helix-turn-helix domain
The first DNA-binding protein motif to be recognized was helix-turn-helix which was originally identified in bacterial proteins. It is constructed from two α helices connected by a short extended chain of amino acids, which constitutes the “turn” The C-terminal helix is called the recognition helix because it fits into the major groove of DNA; its amino acid side chains, which differ from protein to protein, play an important part in recognizing the specific DNA sequence to which the protein binds.
This domain is characteristic of DNA - binding proteins containing a 60- amino acid homeodomain which is encoded by a sequence called the homeobox. In the Antennapedia transcription factor of Drosophila , this domain consists of four α - helices in which helices II and III are at right angles to each other and are separated by a characteristic β - turn.
The characteristic helix -turn helix structure is also found in bacteriophage DNA - binding proteins such as the phage A cro repressor , lac and trp repressors, and cAMP receptor proteins , CRP.
2. Zinc finger domain
The zinc finger domain is generally between 23 and 28 amino acids long and is stabilized by coordinating zinc ions with regularly spaced zinc-coordinating residues (either histidines or cysteines). This domain exists in two forms. The C2H2 zinc finger has a loop of 12 amino acids anchored by two cysteine and two histidine residues that tetrahedrally co-ordinate a zinc ion. This motifs folds into a compact structure comprising two β - strands and one α- helix, the latter binding in the major groove of DNA. The α- helical region contains conserved basic amino acids which are responsible for interacting with the DNA. This structure is repeated nine times in TFIIIA, the RNA Pol III transcription factors. Usually, three or more C2H2 zinc fingers are required for DNA binding.
A related motif, in which the zinc ion is co-ordinated by four cysteine residues, occur in over 100 steroid hormone receptor transcription factors. These factors consist of homo- or hetero- dimers, in which each monomer contains two C4 zinc finger motifs.
3. Leucine zipper
Leucine zipper proteins contain a hydrophobic leucine residue at every seventh position in a region that is often at the C-terminal part of the DNA- binding domain. These leucines lie in an α- helical region and the regular repeat of these residues forms a hydrophobic surface on one side of the α - helix with a leucine every second turn of the helix. These leucines are responsible for dimerizatin through interactions between hydrophobic faces of the α - helices. This interaction forms a coiled - coil structure. bZIP transcription factor contain a basic DNA- binding domain N- terminal to the leucine zipper. This is present on an α - helix which is a continuation from the leucine zipper α - helical C- terimal domain. The N- terminal basic domains of each helix form a symmeterical structure in which each basic domain lies along the DNA in opposite directions, interacting with a symmetrical DNA recognition site so that the protein in effect forms a clamp around the DNA. The leucine zipper is also used as a dimerization domain in proteins that use DNA- binding domains other than the basic domain, including some homeodomain proteins.
4. HELIX- LOOP- HELIX DOMAIN
The overall structure of this domain is similar to the leucine zipper , except that a non helical loop of polypeptide chain separates two α- helices in each monomeric protein. hydrophobic residues on one side of the C-terminal α- helix allow dimerization. this structure is found in the MyoD family of proteins. As with the leucine zipper , the HLH motif is often found adjacent to a basic domain that requires dimerization for DNA- binding. With both basic HLH proteins and bZIP proteins the formation of heterodimers allows much greater diversity and complexity in the transcription factor repertoire.