Chapter 12: Synthesis and Processing of RNA Copyright © Garland Science 2007

Chapter 12:Synthesis and Processing

of RNA

Copyright © Garland Science 2007

Genome expression includes 2 steps

• Initiation of transcription. Assembly of upstream protein complex; this step determines whether a gene should be expressed or not. • Synthesis & processing of RNA. RNA polymerase synthesizes mRNA & subsequently processes or modifies into mature mRNA.

Figure 12.1 Genomes 3 (© Garland Science 2007)

12-1-1. Synthesis bacterial RNA transcripts.

Relatively simpler, only 1 RNA polymerase

1 DNA strand (coding strand) is used as the template

A-U or C-G base pairing; addition of 1 base w/removal of 2 phosphates

Figure 12.2-3 Genomes 3 (© Garland Science 2007)

12-1-1. Cont.

During elongation, RNA polymerase has four subunits, two α (35 kDa) & β+β’ (150 kDa); σ dissociates after transcription initiation.

Occupies 30-bp area (2 transcription bubbles of 12-14 bp & 8 bp RNA-DNA duplex).


Non-template DNA

Template DNA

New RNA

β’ subunit

β subunit

ds DNA

Covalent bonds between amino acids & RNA or DNA (not too tight & not to loose); X-ray crystallography & cross-linking showed RNA synthesis happens between β & β’ subunits; synthesis is not at constant rate; has random pauses <6 msec; causes backtracking.

Figure 12.5 & 7 Genomes 3 (© Garland Science 2007)

12-1-1. Transcription termination

Thermodynamic favors either continuation or dissociation (NOT stop codons)

Two mechanisms: intrinsic terminators (50% of genes, inverted palindrome + poly As); Rho dependent (helicase breaks DNA-RNA base pairs)


12-1-2. Control of termination

Anti-termination by specific proteins; binds upstream of operon; transfers to RNA polymerase; leads to ignorance of termination signals in operon.



Bacterial mRNA is translated at the same time as it is synthesized.

Attenuation as a mean to regulate mRNA synthesis; primarily with operons for amino acid synthesis; results in premature termination.



Attenuation in E. coli tryptophan operon. Ribosome binds at different places & determines formation of large or small RNA loops (the smaller is the termination signal).

Biological significance: When Trp is plenty, attenuation prevents transcription of Trp biosynthesis genes.

Figure 12.12a Genomes 3 (© Garland Science 2007)


Attenuation in Bacillus subtilis tryptophan operon is assisted by trp RNA binding attenuation protein (TRAP); determines formation of large or small RNA loops (termination signals).



Transcript cleavage proteins (GreA & GreB) can reposition the magnesium ion & stimulate RNA polymerase cleavage activity &cut off detached RNA end & prevent stalling of a backtracked polymerase.


12-1-3. Processing of bacterial RNAs

mRNAs of protein-coding genes are ready-to-use in bacteria, but tRNA & rRNA are synthesized as precursors & need cuttings.

3 types of rRNA (5S, 16S, & 23S) by sizes by sedimentation analysis; cut by RNase III, P & F; trim by M16, M23, & M5.


5

4

3

2

1

tRNA processing includes a series of cuttings by different RNases


12-1-3. Processing of bacterial RNAs

Final step is chemical modifications; 50 different types; tRNA modification makes tRNA recognize >1 codons; rRNA is modified by either adding a methyl group to 2’-OH or converting uridine to pseudo-uridine.


12-1-4. Degradation of bacterial RNAs

Transcriptome is responsive to environment & cell physiological status; it constantly changes, therefore, needs degradation, an important way to regulate genome expression.

2 steps: endonuclease cuts off the hairpin; exonuclease degrades from 3’ to 5’. RNases E & P as a protein complex called degradosome


12-2-1. Synthesis of mRNAs in eukaryotes

Chemistry is the same as in bacteria; RNA polymerase II has equivalent α, β, β’ subunits; synthesis is quite different (e.g. poly A tail, 5’ cap, splicing, etc); processing is at the same time with synthesis; Promoter clearance is transition between pre-initiation complex and synthesis; promoter escape RNA polymerase II moves away from promoter.


12-2-1. (Cont.) Initiation5’ capping occurs immediately after initiation. After synthesis of 30 nt, γ- phosphate of 5’ nt is removed; GTP is attached; 7’-N is methylated (type 0 cap); type 1 & 2 caps are also common. Significance: exportation from nucleus & translation

Table 12.1 Genomes 3 (© Garland Science 2007)

12-2-1. (Cont.) Elongation

Bacteria: a few min to synthesize a gene at rate of 100 nt per min; Mammals: a few hours for a gene at a rate of 2000 nt per min; why? Introns (e.g. dystrophin pre-mRNA 2400 kb, 20 hrs to synthesis)

RNA poly II may pause transcription, so it needs elongation factors (proteins associated with polymerase) which can modify chromatin structure.


12-2-1. (Cont.) Termination

3’ poly A tail: addition of up to 250 As by PolyA polymerase in most mRNAs; signals in pre-mRNA are recognized by cleavage & polyA specificity factor (CPSF) & stimulation factor (CstF), polyA binding protein (PADP) helps to recruit As. Significance: mRNA stability & translation.


12-2-1. (Cont.) Regulation of synthesis

3 possible control mechanisms: 1. RNA polymerase elongation factor (TFIIS) can restart a stalled synthesis; 2. Kinases can change phosphorylation status of C terminal domain; 3. alternative polyadenylation (poly A at 3’ ends).


12-2-2. Processing – Remove introns

Most mammalian genes contain 50 or more introns; their positions are sometimes identical in related species (implications in genome evolution); must be excised before it can function as mature mRNA.



Most common introns in vertebrates contain conserved 5’-GU-3’ & 5’-AG-3’ motifs; some also contain a poly-pyrimidine tract as splicing signals.



Splicing in 2 steps: 5’ hydroxyl attack by an internal A & trans-esterification & formation of a loop (lariat structure); 3’ trans-esterification & joins with an adjacent exon. Excised intron is degraded.



Splicing errors: exon skipping (long distance between exons) results in an exon lost in mRNA; cryptic splice site (sequence similarity with the splicing motifs) results in part of an exon lost in mRNA.



snRNAs & proteins (small nuclear ribonucleoproteins, snRNPs) involve in splicing but not completely clear yet: commitment complex; pre-spliceosome complex; spliceosome brings 5’ & 3’ in close proximity & double trans-esterification occur.

Figure 12.32b Genomes 3 (© Garland Science 2007)


Alternative splicing: e.g. 35% of human genes (35,000 or so) have alternative splicing & give 80,000-100,000 different proteins.



Biological significance of alternative splicing: Examples: the human slo gene has 35 exons, 8 are optional; 88=40,320 possible splicing ways but 500 in reality; each specifying a membrane protein with subtle differences; active in inner ear hair cells to detect different sound frequencies 20Hz-20,000Hz (auditory range of human). In fruit fly, alternative splicing determines sex (recommended reading).



Trans-splicing: splicing between different RNA molecules; happens in a few organisms; the leader segment is called spliced leader RNA (SL RNA); plays a role in gene expression regulation.


12-2-3 & 4. Synthesis & Processing of rRNA & tRNA are recommended readings. (p.363-367)


12-2-5. Chemical modification of eukaryotic RNAs

Small nucleolar RNAs involve in pre-rRNA modification at 5th NT of D box.

Many snoRNA are synthesized from intron RNA after splicing.



Chemical modifications can change coding properties & amino acid sequence, called RNA editing.

Biological significance: rare but important; generation of antibody diversity; RNA synthesis; etiology of viral disease.

Above: example of human apolipo-protein (lipid transportation); deamination of a cytosine results in a stop codon & truncated protein.



Other RNA editing: Pan-editing (extensive insertion of nucleotides into abbreviated RNAs to produce functional RNAs); Insertional editing in some viral RNAs; Polyadenylation editing in mitochondrial mRNAs to create termination codon.


12-2-6. Degradation of eukaryotic RNAs

Half life: 10-20 min in yeast & several hrs in mammals. How to control?

Deadenylation-dependent decapping triggered by removal of poly(A) tail; 5’-3’ exonuclease digestion.



Nonsense-mediated RNA decay (NMD) or mRNA surveillance degrades mRNA with an incorrect termination codon (either by mutation or incorrect splicing); again, 5’-3’ degradation.



RNA interference triggered by Dicer ribonuclease & generate short interfering RNAs (siRNAs) of 21-28 bp; degraded by RNA induced silencing complex (RISC); protect host from RNA virus.



microRNA (miRNA) interference is initially synthesis as precursors (foldback RNAs); then cut by Drosha into hairpins; transported into cytoplasm & cut by Dicer; silence mRNAs.

Anneals to 3’ untranslated region of target mRNA & interferes with protein translation.


12-2-6. Transportation of eukaryotic RNAs

80% of total RNA in nucleus need to be transported to cytoplasm for protein synthesis through nuclear pore complexes.

The transportation requires energy (GTP to GDP); 20 different exportins & importins in human; possibly triggered by splicing pathway.

Chapter 12 Summary

Bacterial RNA polymerases synthesize RNAs at a discontinuous rate interspersed by brief pauses due to structural rearrangements; termination can be by 2 mechanisms; functional RNAs are synthesized as precursors & trimed & chemical modified; degradation is controlled by enzymes.

Eukaryotic mRNAs are capped by 7-methylguanosine at 5’ end & poly(A) at 3’ end; Pre-mRNAs contain introns & spliced w/snRNAs; alternative splicing enables more transcripts from one gene; rRNA is chemically modified w/snoRNAs; in mRNA is less common; diverse mechanisms of degradation (e.g. RNA silencing).

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Chapter 12: Synthesis and Processing of RNA Copyright © Garland Science 2007