posttranscriptional modification. eukaryotic mrna modification prok mrna is mature and ready for...

Posttranscriptional Modification

Eukaryotic mRNA modification• Prok mRNA is mature and ready for

translation when it is synthesized

• Euk mRNA requires modification so that it can be translated– Modification at the 5’ end– Modification at the 3’ end– Removal of introns and joining of

exons

Eukaryotic mRNA modification• Euk mRNA requires modification so

that it can be translated– mRNA cannot be exported from the

nucleus until modified– Stabilizes/protects the mRNA from

degradation; rapidly degraded when no 5’ or 3’ modification

– Recognition of mRNA by ribosomes

Eukaryotic mRNA modification• 5’ end modification—5’ capping

– Addition of a ‘cap’ to the 5’ end of the mRNA– Added by capping enzyme– 7-methyl guanosine (m7G)– Attached to the RNA via a 5’-5’ linkage– Nt at position 1 and 2 of the mRNA are

methylated on the sugar group– Prevents degradation – Needed for recognition/binding by the

ribosome

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 11.8 Cap structure at the 5 end of a eukaryotic mRNA

Eukaryotic mRNA modification• 3’ end modification—poly(A) tail

– Addition of a string of adenines to the 3’ end of the mRNA; poly(A) tail

– Usually 50-250 adenines– No template is necessary to add the

tail– Carried out in the nucleus by

enzymes/protein complexes


– Recognizes the poly(A) consensus sequence (5’-AAUAAA-3’)

– Found at the 3’ end in the 3’ UTR– Poly(A) addition site is usually 10-30

nucleotides downstream of the poly (A) consensus sequence

– Since no termination mechanism used during tc, the addition of the poly(A) tail is used to determine the length of the mRNA


– CPSF (cleavage and polyadenylation specificity factor) binds to the poly(A) consensus seq in the newly synthesized pre-mRNA

– CstF (cleavage stimulation factor) binds to a GU- or U-rich region that is downstream to the poly(A) consensus seq in the pre-mRNA

– CPSF and CstF bind to one another; loops the pre-mRNA


– CFI and CF II (CF=cleavage factor) bind pre-mRNA and cleave it at the cleavage site

– PAP (poly(A) polymerase) binds and adds the adenines to the new end of the mRNA; ATP is the substrate

– PABII protein (poly(A) binding protein II) is bound to the poly(A) tail


Fig. 11.9 Diagram of the 3 end formation of mRNA and the addition of the poly(A) tail

Eukaryotic mRNA modification• mRNA splicing

– Euk RNA has introns (intervening sequences)

• Noncoding• Interrupt the coding regions (exons)• Introns are removed• Exons joined back together

– Carried out in the nucleus– Must be completed for mRNA export and

translation


Fig. 11.10 General sequence of steps in the formation of eukaryotic mRNA


– Carried out by spliceosome• complex of proteins and snRNAs• Called small nuclear ribonucleoprotein

particles or snRNP’s (snurps)• 5 main snRNA’s are U1, U2, U4, U5, U6

– Introns are recognized by consensus sequences at the 5’ and 3’ splice junctions

• 5’-GUNNNNN• NNNNNNNNAG-3’


– U1 snRNP binds 5’ splice junction• U1 base pairs with the splice junction—

recognition

– U2 snRNP binds branch point sequence where the 5’ end will bind to form the lariat

• Branch point consensus seq—YNCURA• 5’ binds to the A


– U4/U6 binds to U5; U4/U6/U5 binds to U1 and U2 and RNA is looped to bring junctions close together

– U4 dissociates– snRNPs cleave the intron at 5’ junction

and it is bonded to the A in the branch point sequence—RNA lariat is formed

– 3’ junction is cleaved and the 2 exons are covalently joined together.


Fig. 5.12 Details of intron removal from a pre-mRNA molecule


Fig. 11.11 Model for intron removal by the spliceosome

Ribosome structure and rRNA• Ribosomes composed of 2 subunits

– 1 large subunit and 1 small subunit• Proks (E. coli): large subunit=50S and small

subunit=30S; together they are 70S

• Euks (mammals): large subunit=60S and small subunit=40S; together they are 80S

– Subunits are composed of many proteins and at least 1 rRNA; rRNA is catalytic

– Euk ribosomes are larger and more complex than prok ribosomes


Fig. 5.16 Composition of whole ribosomes and of ribosomal subunits in mammalian

cells

Transcription of rRNAs--E. coli • rRNA genes are arranged together

in the DNA to form transcription units

• rRNA genes have some tRNA genes embedded in the transcription unit

• Called rrn region; 7 of these regions in the E. coli genome

Transcription of rRNAs--E. coli • The rRNA is transcribed as one

large piece—precursor rRNA (pre-rRNA)

• Cleaved into mature rRNAs (16S, 23S, 5S, and tRNAs) by RNase III and other enzymes

• Associate with ribosomal proteins as transcription is occurring.


Fig. 5.17 rRNA genes and rRNa production in E. coli

Transcription of rRNAs—Euks • rRNA genes are arranged together

and repeated in tandem in the DNA 100-1000 times

• Transcription at these regions produces the nucleolus (site of ribosomes assembly)

• All but 5S rRNA are synthesized from this gene cluster; 5S is located elsewhere in the genome

Transcription of rRNAs—Euks • rRNA is transcribed by RNA polymerase I

– Requires tc factors to bind to the DNA – Requires a promoter– Termination mediated by termination seq

• The rRNA is transcribed as one large piece—precursor rRNA (pre-rRNA)

• Cleaved into mature rRNAs (18S, 5.8S, 28S)

• Associate with ribosomal proteins and assembled into ribosomes in nucleolus.


Fig. 5.18 rRNA genes and rRNA production in eukaryotes

Transcription of tRNAs—Euks • Carried out by RNA polymerase III

– Tc tRNAs, 5S rRNA, and some snRNAs– Require tc factors to bind to DNA and

promoter

• tRNA genes are repeated in the euk genome

• Each tRNA is unique but all have CCA added to 3’ end and are extensively modified posttranscriptionally

Transcription of tRNAs—Euks • tRNAs undergo extensive secondary

structure—cloverleaf structure

• Contains an anticodon that is complementary to the codon in mRNA.

• Some tRNAs have introns that must be removed by splicing.


Fig. 5.21 Cloverleaf structure of yeast alanine tRNA


Fig. 5.19 Transcription factors involved in the initiation of human rDNA transcription

by RNA polymerase I


Fig. 5.22a Three-dimensional structure of yeast phenylalanine tRNA as determined

by X-ray diffraction of tRNA crystals


Fig. 5.24 Cloverleaf models for yeast precursor tRNA.Tyr and mature tRNA.Tyr


Fig. 5.1 Transcription process

posttranscriptional modification. eukaryotic mrna modification prok mrna is mature and ready for...

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