Chapter 26

RNA Metabolism
DURRIYA NAEEM KHAN
Topics to cover:

 DNA-DEPENDENT RNA SYNTHESIS

 RNA PROCESSING

 RNA-DEPENDENT SYNTHESIS OF RNA and DNA

General Concepts in TRANSCRIPTION:
 Transcription
 mRNAs
 Transfer RNAs
 Ribosomal RNAs
 Transcriptome
 Template strand
 Non-template strand
 Which strand is coding strand?
 How it is determined which region is going for the transcription?
 Consensus sequences?
 Promoters?
DNA dependent RNA synthesis
 3phases?
 Initiation, elongation and termination
 How it differs from Replication??
 No primer, only one strand and specific region
 Polymerase?
 DNA-dependent RNA polymerase.
 Four nucleotides?
 Other factors?
 Magnesium and Zinc.
 Direction of synthesis?
 RNA pol binds tightly to dsDNA but only one strand serve as template.
 Base pairing different
A=T G=_C
 Initiation starts when RNA pol binds to DNA at???
 Promoter
 ( DNA-RNA hybrid of about 8bp leads to the dissociation of transcript from the DNA.
 At any given time 17bp is unwound on DNA.
 Elongation rate 50-90nts/s
 RNA POLYMERASE
 5 CORE SUBUNITS AND A σ SUBUNIT
 σ SUBUNIT DETERMINES THE PROMOTER SEQUENCES.
 It binds to the core subunits and directs the enzyme to the specific binding sites.
 Many types of σ subunit are present acc to their size – differentiated
 Most commonly studies----σ70
 No proofreading activity 3’-5’ site in pol.
 But RNA pol and RNA poI II in eukaryotes do halt when mismatch resulted and removes it from 3’ end. (direct reversal
of polymerization)
PROMOTERS:
 RNA pol binds to specific sequence called promoters.
 Promoter region extends from 70bp before start site to 30 bp beyond the start site
 -70 to +30bp
 σ70 recognizes -10 and -35 centered sequences.
 All promoter sequences are not similar but certain nucleotides that are common called the consensus sequences.
 Consensus sequence at -10 is 5’ TATAAT 3’
 Consensus sequence at -35 is 5’ TTGACA 3’
 A=T rich recognition element called UP( upstream promoter)elements occurs between positions -40 and -60 .
 UP element is bound by α subunit of pol.
 Mutation in consensus sequences leads to?
 INITIATION:---- 2PARTS
 1. BINDING 2.INITIATION
1.BINDING
 Polymerase attaches to the DNA by ????
 Two complexes are formed: open and closed
 Closed Complex: in which the bound DNA is intact
 Open complex: in which the bound DNA is intact and partially unwound near
-10sequence.
 Promoter Clearance: conformational change in the complex leads to elongation and
movement away from the promoter.
σ70 dissociates from the region and then

NusA proteins bind to the elongating RNA pol competitively with the σ70

Other classes of σ
σ32 factor is for Heat shock protein promoters.
TRANSCRIPTION REGULATION
 Regulation can occur at any step in transcription.
 Mostly directed at polymerase binding and initiation.
 Protein binding can affect the transcription. E.g
 Transcription activator: cAMP receptor protein (CRP). Increases the transcription of genes.
 Repressors proteins blocks the synthesis of RNA.

 SIGNAL TERMINATION of RNA synthesis:

 Certain DNA sequences pause the RNApol to further transcribe the strand
 E.coli: two classes of Termination signals.
 A- RHO DEPENDENT
 B- RHO INDEPENDENT.

 RHO IS??
 PROTEIN FACTOR.
ρ INDependent termination
 Transcript have self complementary sequences that leads to the formation of???
 Hairpin structure. (15-20nts before the end)
 String of three A residues at end of the 3’ end
 Hairpin structure disrupt the association of DNA-RNA or RNA and RNA pol or both
resulting in isomerization. A=U hybrid at 3’ end is relatively unstable that leads to the
dissociation of RNA from the complex.
 if no A=U bases that WHAT WILL HAPPEN?

 ρ Dependent termination
 Lacks A string
 Have C-A rich sequence called rut (rho utilization element)
 Rho protein encounters RNA and then travel down the road and say hi to the complex at
the end of 3’ and make it realize you have to dissociate from the strand and say, no
offense strand does not want you any more.
 It’s a ATP dependent RNA-DNA HELICASE activity. Benefit???? Why the helicase activity?
Eukaryotes: Nuclear polymerase: 3
kinds
 RNA pol. I, II, III

 RNA pol I
 synthesis of pre rRNAs
 contain precursors of 18S, 5.8S,28S rRNAs
 its promoter differ greatly in sequence across species.
 RNA pol II
 synthesis of mRNAs and some specialized RNAs
 recognize variety of promoters that vary in sequence
 some promoters have common features like TATA box near -30 bp or Inr
sequence near start site+1
 RNA pol III
 synthesis of tRNAs and 5S rRNAs and some other small specialized RNAs
 RNA pol II
 Huge enzyme– 12subunits.
 Largest subunit called ---- RBP1 homologous to bacterial β’ subunit of RNApol(α 2 ββ’ωσ)
 Subunit RBP2 structurally similar to β subunit of bacterial RNA pol
 RBP3 and RBP11 also structurally similar to α subunit.

 RBP1 also has a long C-terminal tail having many repeats of heptad amino acid
-YSPTSPS-
 27 REPEATS IN YEAST.
 52 in human.
 CTD (carboxy terminal domain) separated from main body enzyme through unstructured
linker sequence.
Proteins in Transcription complex
formation
 Called transcription factors

 Highly conserved in all eukaryotes.

 FACTORS: involves in preinitiation complex(closed complex to open complex)

- +1
Its bounds the
TBP and also 30
TATA Inr
itself binds to Binds to the TATA
DNA box. It’s a part of a
complex called TFIID
TBP
TFIIA TFIIB
TFIIF and
Pol II
Its binds to TFIIE TFIIH
help
stabilize the
TBP DNA Helicase activity; unwind DNA Binds to the TFIIB-TBP complex.
complex at start site; making it open TFIIF helps binding of pol to its
complex promoter.
 RNA strand Initiation and PROMOTER
CLEARANCE
TFIIH KINASE ACTIVITY------??

 Phosphorylates: CTD of RNA pol II

 Other kinases :::::: CDK9 (part of complex pTEFb[positive transcription elongation factor
b)
also phosphorylates the CTD repeat seq.(Ser residues)

 Conformational change--- transcription starts.

 Initial synthesis of 60-70 nts renders the removal of TFIIE and then TFIIH is released and
now the state enters in ELONGATION; VOILA.
Elongation, Termination and Release
 TFIIF always with Pol II throughout elongation
 ELONGATION FACTORS: increase pol activity.
 Few binds to PHOSPHORYLATED CTD suppressing the pause during transcription.

 Termination Factors helps in terminating the process by dephosphorylating CTD and

dissociation of EF factors.

 TFIIH: some components involve in Nucleotide excision repair complex.

 Loss of TFIIH : genetic disease XENODERMA PIGMENTOSUM

 COCKAYNE Syndrome.
POLYMERASE INHIBITION: Actinomycin D: in both bac and eukary: interclate between G=-
Cbp and halts the template movement. Also inhibits elongation.
Acridine
Rifampicin--- bacterial RNA synthesis by binding to beta subunit preventing promoter
clearance.
 Mushroom -----produces α-AMANITIN
 BLOCKS RNA SYNTHESIS IN EUKARYOTES
 POL I IS INSENSITIVE TO IT .
RNA PROCESSING
 General concepts
 RNA processing?
 In eukaryotes or prokaryotes?
 Ribozyme?
 Primary Transcript?
 RNA splicing?
 5’CAP?
 Poly ‘A’ tail?
CAPPING AT 5’ END
7 methyl guanosine at 5’terminal residue of mRNA through 5’,5’-triphosphate linkage.

Helps in protection from RIBONUCLEASES.

Also recruits special cap binding proteins to initiate translation.

CAPPING enzymes
Phosphohydrolase
BINDS WITH CTD
guanylytransferase
guanine-7-methyltransferase
CBC-------CAP BINDING COMPLEX.
SPLICING:
 Four classes of introns
 Group I and group II introns. they differ completely except they are SELF-SPLICING
means no proteins or enzymes are involved in it.
 GROUP I INTRONS: code for rRNAs , tRNAs and mRNAs.
 GROUP II INTRONS: in all primary transcript of MTCH and CHLOROPLAST in plants
fungus algae.
 no ATP requirement
 Transesterification reaction for splicing

Group I introns require Guanine nucleoside or nucleotide as a nucleophile not as

source of energy.
Group II introns uses 2’OH group of A residue of intron. A Lariat structure is formed as
an intermediate.
Spliceosomal Introns
 Largest group of introns found in primary transcripts.
 Spliceosomal Introns are removed by large protein complex called SPLICEOSOME.
 Splicing occurs within spliceosome by mechanism of group II introns.

 SPLICEOSOME:
 made up by specialized protein complexes called small nuclear
ribonucleoproteins (snRNPs or snurfs)
 Snurfs contain 100-200nt long RNA called SMALL NUCLEAR RNAs( snRNAs)
 Five snRNAs– U1, U2, U4, U5 and U6. involved in splicing.
 Splicing occurs in which compartment?????

 Tightly regulated with transcription.

 four group of introns require ATP. FOUND IN CERTAIN tTRNAs
3’ END OF mRNA
 Poly A tail 20-250 A residues
 Poly A tail and its associated proteins helps in protection of mRNA from enzymatic
degradation
 In bacteria--- signal of degradation

 Mechanism of addition of poly A tail

 transcription extended beyond the poly A addition site.
 Endonuclease of CTD cleaved the Poly A addition site.
 This cleavage is marked by two sequence
 5’ AAUAAA3’ Highly conserved 10-30 nts upstream of cleavage site
 rich in G and U residues 20-40nts DOWNSTREAM of cleavage site.

 Cleavage results in FREE 3’OH group to which A residues are added by

POLYADENYLATE POLYMERASE.