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

Fig. 11.8 Cap structure at the 5 end of a eukaryotic mRNA Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

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

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

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

Eukaryotic mRNA modification 3’ end modification—poly(A) tail 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 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

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

Eukaryotic mRNA modification mRNA splicing 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’

Eukaryotic mRNA modification mRNA splicing 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

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

Fig. 11.11 Model for intron removal by the spliceosome Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

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

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

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

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

Fig. 5.19 Transcription factors involved in the initiation of human rDNA transcription by RNA polymerase I Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 5.22a Three-dimensional structure of yeast phenylalanine tRNA as determined by X-ray diffraction of tRNA crystals Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 5. 24 Cloverleaf models for yeast precursor tRNA Fig. 5.24 Cloverleaf models for yeast precursor tRNA.Tyr and mature tRNA.Tyr Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 5.1 Transcription process Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.