transcriptron.pptx - Slide 1

RNA synthesis:transcrition

• Transcription is the process of the synthesis RNA molecule.

Transcription unit

• Promoter region• Transcribed region• And termination region.

Promoter region

• Which is certain sequence of base located at the beginning of stretch. It is important for initiation for transcription to occur and can be recognized by RNA polymerase.

prokaryotes

• 1. The pribno box :stretch of 6 nucleotides (TATAAT) located about 10 bases to the left of the transcription. Initiation site.

• 2. A second nucleotide stretch (TGTTGACA) that located about 35 bases to the left of the transcription initiation site.

• 3. 19 bases (nucleotide) in between two streches.

Cont.

• Both pribno box and TGTTGACA are region can be recognized by RNA polymerase.

Eukaryotic promotor

• 1. The Hoges or TATA box: stretch of nucleotide that is almost similar to prokaryotes,located almost 25 nucleotide to the left of the trancrition initiation site.

• 2. CAAT box : located about 70 to 80 nucleotides to the left of the trasncription initiation site.

• 3. 40 bases between the two stretches.

Cont.

• Transcription region: which is stretch of DNA that is to be transcribed into RNA molecules.

• Termination region: which is a stretch of DNA located at the end of DNA sequence to be trasncribed.

Requirement for RNA synthesis

• Transcription unit: transcription occurs on one of the two strands of the DNA template and never on both complementary strand

• Four ribonucleotide triphosphate;ATP,GTP,UTP and CTP.

• RNA poymerase ,RNA(=DNA –dependent RNA polymerase)

Prokaryotes RNA polymerase.

• 1.E.g E.coli ,enzyme synthesize all types of RNA molecules on DNA template.

• 2. the holoenzyme of the RNA polymerase consist of a core molecule and specific protein factor(sigma [σ] factor.

• A. The core enzyme molecule : 4 subunits: 2 of them are identical (alpha subunits.the other 2(β and β’) are similar but not identical .RNAP consist of 2 zinc molecules.

Sigma factor

• Enables the polymerase to recognize promotor regions on DNA .

• Helps the core enzyme to attach more tightly to the promotor site.

• Some regions on DNA that signal the termination of transcription are recognized by the RNA polymerase itself. Others are recognized by specific termination factor e.g rho factor of E.coli.

Steps RNA synthesis

• Initiation • Elongation• termination

Initiation

• 1. RNA polymerase holoenzyme binds with the promotor area:

• A . Sigma subunit ,enables polymerase polymerase to recognize promotor region on DNA.

• B. Β̒�subuint to the template.• C. B subunit binds to the nucleotide substrate.

Cont.

• 2. binding of RNA poymearse DNA template leads to local separation (unwinding)of the DNA double helix into sense and antisense strands.

• 3. first nucleotide of RNA transcript at the initiation site is almost always purine.

Elongations

• 1.At the sense formation of RNA molecule begins at the end of core enzyme with release of sigma 5̒ 5̒�

factor.• 2. Then elongation of the RNA molecule occurs

from 5 to end ,antiparallel to its template. ̒3̒ ̒� 3̒�

• 3. nucleotide building blocks are ribonucleotide 5̒ 5̒�

triphosphate(ATP ,GTP,CTP,and UTP). They are inserted in the RNA = pairing rule.pyrophosphate is released when each new nucleotide is added to growing chain . GTPGMP + PPi

Cont.

• 4.RNA polymerase forms a phosphodiester bond between the ‘3” OH of one ribose sugar and 5’ OH of the next ribose.

• 5. the process of elongation of RNA chain continues until a termination region reached.

Termination:

• Termination region on DNA template can be recognized by :

• A. RNA – polymerase enzyme itself (rho independent termination),.

• B. Rho [ρ] factor which may be required for the release of both RNA strand and RNA polymerase (rho dependant termination).

Termination result from either

• A. Binding of rho factor polymerase enzyme : when RNA – polymerase enzyme reaches the termination site,rho factor binds with it causing termination.

• Slowing down of RNA – polymerase at the termination site:

• 1. termination site on DNA is charaterized by the presence of palidromes.

Cont.

• Palidromes is a region of a double stranded DNA in which each of the two strands haas the same sequence when read in the same direction. E.g in the 5’ to 3’direction.

• 2.The RNA transcript of the DNA palidrome can form a stable hairpin structure which is self –complementary structure. This hairpin structure causes slowing of RNA – poylmerase at the termination site.

antibiotics

• Rifampcin: binds to the beta subunit . Treatment of leprosy and TB patients.

• Actinomycin D: binds to DNA template ,prevent movement of RNA – polymerase along the DNA.

• Alpha –amanitin: toxin from mushroom , inactivates RNA – polymerase II of eukaryotes.

Post transcriptional modification of RNA

• A. eukaryotes mRNA : primary mRNAs are called heterogeneous nuclear RNA (hnRNA) .hnRNA is modified into mature mRNA in the nucleus by capping additon of nucleotides and splicing.

5’capping

• The 5’end of the RNA requires a cap which is 7 methyl-guanosine triphosphate. It is attached by a 5’to 5’triphosphate linkage.

• A. reaction needs an enzyme called guanyl transferase.

• B. function of this cap is to facilitate the initiation of translation, and protects the 5’end of mRNA from attack by 5’to 3’exonucleases.

Addition of poly (A) tail.

• mRNA require almost 40 to 200 adenine nucleotides added at 3’terminus to form a poly adenine (A) tail:

• poly A polymerase . Enzyme for the reaction.• This tail protects the 3’end of mRNA form 3’to

5’ exonuclease attack.

Splicing (removal of introns)

• hnRNA is formed many pieces of some of them (exons) will be translated into amino acids.

• Others (introns) will be not be translated into amino acids and must be removed before translation takes place.

spliceosomes

• Responsible for removal of introns from the hnRNA , and splicing (ligation) of both ends of exons to form mature mRNA.

• It consist of primary hnRNA ,4 small nuclear RNA’s (U1,U2,U5, and U4/U6) and an undetermined number of proteins.

• Smaller nuclear RNA’s (snRNA) is to bind each end of the introns by forming base pair with each other.

cont.

• Spliceosomes facilitate also the transport of mature mRNA from the nucleus to the cytoplasm.

• Defect in the process of spilicing may lead to disease e.g at least one from of beta thalassemia , a disease in which there is absent synthesis of beta chain of hemoglobin appears to result from a nucleotide change at an exon-introns junction.

Transfer RNA(t RNA)

• 1.It serves as a adapter mol. For the translation of mRNA into protein sequence.

• 2. primary tRNA transcript are subjected to many modifications:

• A. tRNA mol are transcribed as larger precusors. These precursors are reduced in size by specific class of ribonucleases.

• B. Attachment of the characteristics C.C.A terminus at the 3’end of the molecules.

Cont.

• The tRNAs contain many modification of the standard bases A,U,G and C .Some bases are methylated, alkylated, or attached to carbohydrate residue by glucosidic bonds.

• some tRNA contain near to the anticodon loop a single intron 10-40 nucleotides long. These introns are removed with splicing ofr exons to produce an active tRNA for protein synthesis.

Ribosomal RNA(rRNA)

• 1.In mammals cells ;rRNA is transcribed as a single larger precursor molecules called 45s.

• 2.In the nucleus ,45s is methylated and cleaved by specific endonucleases and exonucleases to give four kind of r RNA : 5SrRNA,5.8SrRNA,18SrRNA and 28SrRNA.

Cont.

• 3. The four kind rRNA combine with a number proteins to form a ribosomes.

• 1. ribosomes are cytoplasmic nucleoproteins composed of 4 rRNAs plus a number of proteins.

• 2. These rRNAs and proteins are distributed specifically between the two smaller and larger ribosomal subuints.

Cont.

• 3. The smaller subunits is called 40S .It contains one 18S rRNA and 33 proteins.

• 4. The larger subunit is called 60S . It contains the remaining r RNAs (28S,5.8S and 5S) and 45 proteins.

• 5. Both smaller (40S) subuint form the whole 80S ribosome.

Function of ribosomes

• They are site of protein synthesis within cells.