《分子生物学 Molecular Biology》课程教学课件（PPT讲稿，英文版）Chapter 15 Messenger RNA Processing II - Capping and Polyadenylation

Copyright@The McGraw-Hill Companies,Inc.Permission required for reproduction or display. Chapter 15 Messenger RNA Processing ll: Capping and Polyadenylation K.G.Murti/Visuals Unlimited

Chapter 15 Messenger RNA Processing II: Capping and Polyadenylation

15.1 Capping By 1974,mRNA from a variety of eukaryotic species and viruses were found to be methylated(甲基化的) A significant amount of this methylation was clustered at the 5'-end of mRNA This methylation cluster formed a structure we call a cap 15-2

15-2 15.1 Capping ✓ By 1974, mRNA from a variety of eukaryotic species and viruses were found to be methylated (甲基化的) ✓ A significant amount of this methylation was clustered at the 5’-end of mRNA ✓ This methylation cluster formed a structure we call a cap

Cap Structure Early study used viral mRNA as they are easier to purify and investigate The B-phosphate of a nucleoside triphosphate remains only in the first nucleotide in an RNA -Cap is at the 5'-terminus of RNA -The cap is made of 7-methylguanosine(7- 甲基化鸟甘酸，m7G) -Linkage is a triphosphate(三磷酸盐键) Charge on the cap area is near-5 15-3

15-3 Cap Structure ✓ Early study used viral mRNA as they are easier to purify and investigate ✓ The -phosphate of a nucleoside triphosphate remains only in the first nucleotide in an RNA – Cap is at the 5’-terminus of RNA – The cap is made of 7-methylguanosine(7- 甲基化鸟甘酸，m7G) – Linkage is a triphosphate (三磷酸盐键) – Charge on the cap area is near -5

CHa Charges +1 Reovirus Cap Structure √The m7G contributes a positive charge OH OH Triphosphate linkage contributes 3 negative charges √Phosphodiester bond contributes 1 negative charge 0 √Terminal phosphate contributes 2 negative O OH(Methlyated in cap 2) charges -2 15-4 Net:-5

15-4 Reovirus Cap Structure ✓ The m7G contributes a positive charge ✓ Triphosphate linkage contributes 3 negative charges ✓ Phosphodiester bond contributes 1 negative charge ✓ Terminal phosphate contributes 2 negative charges

Cap Synthesis First step pppXpY. RNA triphosphatase -RNA triphosphatase(核苷磷酸 (a ppXpY-. 水解酶)removes terminal Gppp一 phosphate from pre-mRNA (b) Guanylyltransferase -Then,guanylyl transferase( GpppXpY. AdoMet- 苷酸转移酶)adds capping GMP (c) Methyltransferase from GTP m7GpppXpY. √Next,2 methyl transferases(甲 AdoMet- 基转移酶)methylate N7of (d) Methyltransferase capping guanosine and 2'-0- m7Gpppx"pY. B methyl group of penultimate (e) nucleotide CH2 This occurs early in transcription,before chain is OHOH 30 nt long S-腺苷甲硫氨酸（AdoMet) 15-5

15-5 Cap Synthesis ✓ This occurs early in transcription, before chain is 30 nt long ✓First step –RNA triphosphatase (核苷磷酸 水解酶) removes terminal phosphate from pre-mRNA –Then, guanylyl transferase (鸟 苷酸转移酶) adds capping GMP from GTP ✓ Next, 2 methyl transferases (甲 基转移酶) methylate N7 of capping guanosine and 2’-O￾methyl group of penultimate nucleotide S-腺苷甲硫氨酸（AdoMet）

Functions of Caps Caps serve at least four functions: -Protect mRNAs from degradation Enhance translatability(可译性)of mRNAs Transport of mRNAs out of nucleus -Efficiency of splicing mRNAs 15-6

15-6 Functions of Caps Caps serve at least four functions: – Protect mRNAs from degradation – Enhance translatability (可译性) of mRNAs – Transport of mRNAs out of nucleus – Efficiency of splicing mRNAs

15.2 Polyadenylation(多聚腺苷酸化) The process of adding poly(A)to RNA is called polyadenylation A long chain of AMP residues is called poly (A) Heterogeneous nuclear mRNA is a precursor to mRNA Most eukaryotic mRNAs and their precursors have a chain of AMP residues about 250 nt long at their 3'-ends Poly(A)is added posttranscriptionally by poly(A) polymerase 15-7

15-7 15.2 Polyadenylation (多聚腺苷酸化) ✓ The process of adding poly(A) to RNA is called polyadenylation ✓ A long chain of AMP residues is called poly (A) ✓ Heterogeneous nuclear mRNA is a precursor to mRNA ✓ Most eukaryotic mRNAs and their precursors have a chain of AMP residues about 250 nt long at their 3’-ends ✓ Poly(A) is added posttranscriptionally by poly(A) polymerase

Functions of Poly(A) Poly(A)enhances both the lifetime and translatability of mRNA Relative importance of these two effects seems to vary from one system to another √In rabbit reticulocyte(网织红细胞)extracts, poly(A)seems to enhance translatability by helping to recruit mRNA to polysomes 15-8

15-8 Functions of Poly(A) ✓ Poly(A) enhances both the lifetime and translatability of mRNA ✓ Relative importance of these two effects seems to vary from one system to another ✓ In rabbit reticulocyte (网织红细胞) extracts, poly(A) seems to enhance translatability by helping to recruit mRNA to polysomes

Basic Mechanism of Polyadenylation Transcription of eukaryotic genes extends beyond the polyadenylation site √The transcript is: -Cleaved -Polyadenylated at 3'-end created by cleavage 5 (a) Cut Polyadenylate (c) Degrade 15-9

15-9 Basic Mechanism of Polyadenylation ✓ Transcription of eukaryotic genes extends beyond the polyadenylation site ✓ The transcript is: – Cleaved – Polyadenylated at 3’-end created by cleavage

Synthesis and Polyadenylation Copyright The McGraw-Hill Companies.Inc. Permission required for reproduction or display (a) Transcribe,polyadenylate,and splice. + + (b) Transcribe,terminate. Polyadenylate ■■ 2 Transcribe. Clip,polyadenylate. 15-10

15-10 Synthesis and Polyadenylation