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 26| RNA Metabolism

© 2017 W. H. Freeman and Company

 CHAPTER 26
RNA METABOLISM

26.2 RNA Processing

26.3 RNA-Dependent Synthesis of RNA and DNA

 BVC201 Genetics Objectives
9. Draw the complete structures of all nucleotides and short nucleic acid molecules.
10. Describe the structure of DNA and the types of interactions stabilizing the
structure of nucleic acids.
11. Describe the structure of genes within the chromosome.
12. Describe in outline the processes of transcription,
translation, and DNA replication.
13. Know the architecture of genomes and the functions of genetic elements.
14. Explain the mechanisms used to regulate gene expression and use specific
examples to illustrate.
15. Show good knowledge of certain important analytical methods in biochemistry,
and the calculations associated with these methods, as well as a good degree of
laboratory skill when applying these methods within the biochemistry practical
sessions.
 RNA Processing
▪ Newly synthesised RNA molecule = primary transcript
▪ Many bacterial RNA and eukaryotic RNA molecules
processed after synthesis
▪ Ribozymes catalyse most of these post synthetic
processing
▪ The most extensively processing of primary transcripts
occur in:
▪ Eukaryote mRNA
▪ tRNAs in both prokaryotes and eukaryotes
▪ Some special function RNAs

▪ The fate of all RNA is its complete and regulated

degradation
 RNA Processing
What processing can an eukaryotic primary transcript
undergo?
1. 5’-capping.
2. Polyadenylation.
3. Intron splicing:
▪ Self splicing of Group 1 and Group II introns
▪ Splicing with the aid of RNA-protein complexes –
Spliceosomal introns
▪ Spicing by protein enzymes – Splicing Endonuclease
▪ Alternative splicing
4. Cleavage.
5. Editing.
6. Regulated turnover and degradation
 Processing of the ends
of eukaryotic mRNA
1. 5’ cap
▪ protects RNA from ribonucleases
▪ forms a binding site for ribosome

2. 3’ poly (A) tail

▪ string of 30 A residues in yeast and 50-100 in animals
▪ protects 3’ end from degradation
 5’ capping of mRNA
▪ Takes place co-transcriptionally
(early in transcription).

▪ Enzymes in the cap-synthesising

complex and the 5’ end of
transcript associated with
Carboxy-Terminal Domain (CTD) of
Pol II until cap is synthesized

▪ Capped 5’ end is released from

cap-synthesizing complex and
bound by Cap Binding Complex
(CBC) until end of transcription
5’ capping on mRNA

7-methylguanosine
links to 5’-end via 5’,5’-
triphosphate link

Additional methyl
groups are often added
at the 2’ hydroxyls of the
1st and 2nd nucleotides
adjacent to the cap

Fig 26.12; p1071

 Polyadenylation
▪ RNA Pol II synthesizes RNA beyond the
site where poly(A) tail needs to be added
▪ Endonuclease cleave primary transcript
at cleavage site
▪ Cleavage site marked by sequences:
▪ highly conserved 5’ AAUAAA3’ 10- 30 nt
on the 5’ side (upstream)
▪ G and U rich region 20-40 nt
downstream
AUG UGA
5’ 3’
AAUAAA
5’cap
Cleave site
▪ Endonuclease part of large enzyme
complex associated with CTD
▪ Polyadenylate polymerase synthesizes a
poly(A) tail 80−250 nt long beginning at the
cleavage site
 mRNA Processing
▪ mRNA primary transcripts newly
synthesized RNA molecule
including introns and exons
▪ mRNA can undergo:
▪ 5’-capping.
▪ Polyadenylation.
▪ Intron splicing.
▪ Coordinated processing on
primary transcript as it is
synthesised in nucleus, before
export to cytoplasm
▪ Processing is coupled to transport
Figure 26.11
to cytoplasm and delivery to
ribosome for translation
 Intron Splicing
Generally in Prokaryotes:
▪ DNA sequence is collinear with mRNA sequence and polypeptide chain
and amino acid sequence

Generally in eukaryotes
▪ DNA sequence is transcribed continuously, including introns and exons in
the RNA primary transcript

▪ Introns must be removed from the RNA primary transcript

▪ Boundaries of the introns are marked by specific sequences
 Four Classes of Introns
▪ Group I and Group II introns are self-splicing
▪ Require no additional proteins or ATP
▪ In nuclear, mitochondrial, and chloroplast genomes
▪ Group I & II differ mainly in the splicing mechanism
▪ Spliceosomal introns are spliced by enormous RNA-
protein complexes called spliceosomes
▪ The most common introns
▪ Frequent in protein-coding regions of eukaryotic genomes
▪ tRNA introns are spliced by protein-based enzymes
▪ Primary transcript cleaved by endonuclease
▪ Exons are joined by ATP-dependent ligase
 Splicing of Group I Introns
What is spliced ?
▪ Nuclear, mitochondrial and
chloroplast genes coding for rRNA,
mRNA, tRNA
What is required ?
▪ A guanine nucleoside/ nucleotide
cofactor (GMP, GDP/ GTP)
▪ 3’ OH group of guanosine is used as
a nucleophile
▪ Guanosine break the exon-intron
boundary by forming a 3’,5’
phosphodiester bond with the 5’ end
of the intron
▪ The free 3’ OH group of the displaced
exon then acts as nucleophile to
attack the 3’end of the intron
▪ Intron is removed with an G attached Figure 26.14
to 5’ end and the exons are ligated
 Splicing of Group II Introns
What is spliced ?
▪ Mitochondrial or chloroplast
mRNA in fungi, algae and plants
What is required ?
▪ 2’-OH of an A residue within
the intron to act as
nucleophile
▪ Displaces the 3’OH of the
nucleoside at the upstream
exon-intron boundary
▪ A branched lariat-like
intermediate is formed
▪ Free 3’OH attacks the
downstream intron-exon
boundary Figure 26.13
 Spliceosome Introns
▪ What is spliced ?
▪ mRNA (most common method in eukaryotes)
▪ not self-splicing

▪ What is required ?
▪ Specialized RNA protein complexes – small nuclear
ribonucleoproteins (snRNPs) –
▪ Each snRNP contains class of eukaryotic RNA - small
nuclear RNAs (snRNA) (100-200bp)
▪ 5 snRNAs commonly used: U1, U2, U4, U5, U6
▪ Spliceosomal introns generally have GU at 5’-end and AG at
3’-end
▪ These sequences mark sites of splicing
 Spliceosome Introns
▪ The RNA components of the spliceosome catalyst of splicing
steps
▪ The U1 snRNA contain regions complementary to 5’ splice site
of nuclear mRNA
▪ U1 snRNP binds to this 5’ region in primary transcript
▪ U2 snRNP binds to the 3’end
▪ U1 and U2 binding creates a bulge that partly displaces and
activates an A to be a better nucleophile
▪ Next, U4, U5, and U6 bind, bringing at least 50 proteins to create
spliceosome
▪ ATP required for assembly of spliceosome but NOT for RNA
cleavage-ligation reactions
▪ Some parts attached to CTD (carboxy-terminal domain) of RNA Pol II
▪ Indicates coordination of splicing with transcription Figure 26.16
▪ Intron remains in nucleus and is eventually degraded
 Fourth class of introns

▪ What is spliced ?
▪ Introns found in certain tRNA molecules.

▪ What is required ?
▪ The splicing reaction requires ATP &
▪ Endonucleases
▪ Splicing endonuclease cleave at exon-intron borders.
▪ Exons are ligated using ligase reaction.
 Intron Splicing

Intron splice
Splicing Type RNA product Cofactor Catalytic Agent
product

nucleus,
Type I – self mitochondria, 3’OH of external
Ribozyme Open
splicing chloroplasts G
(rRNA, tRNA, mRNA)
mRNA in
Type II – self mitochondria, 2’OH of internal
Ribozyme Lariat
splicing chloroplasts A
(fungi, algae, plants)

2’OH of internal
Spliceosome mRNA in nucleus snRNA Lariat
A

Endonuclease
Endonuclease tRNA None Open
(ATP-dependent)

Note cellular location!

Summary of mRNA
Processing

Figure 26.18
 Can a single gene encode
MORE than one Protein
▪ Historically biochemists believed in a one-gene, one-
protein theory. This was revised to the one-gene,
one-polypeptide theory.

▪ Q : But can a single gene encode MORE than one

polypeptide?

▪ A: Yes. Some mRNAs yield multiple gene products.

 Differential mRNA
Processing

Some mRNAs yield multiple gene products by

two mechanisms:

1. Multiple polyadenylation signals.

2. Alternative splice sites.
 Alternative pre-mRNA processing
Alternative splicing
▪ Different mature mRNAs are
produced from the same primary
transcript.
▪ Alternative mature mRNA
produce different proteins
▪ Alternative splicing patterns:
▪ Different 3’splice sites
▪ Different 5’splice sites
▪ Splice sites can exclude an exon
▪ Splice sites can add an exon

Poly (A) site choice

▪ Complex transcripts have more
than one site where poly (A) tails
can form
▪ Two or more sites for cleavage and
polyadenylation can remove more or
Figure 26.19 a & b less of primary transcript sequence
 rRNA Processing in Bacteria
▪ rRNA transcribed as a single large 30S precursor (6 500 nt)
▪ 5S,16S and 23S and a few tRNA’s are all derived from 30S RNA
precursor
▪ 30 S precursor cleaved to mature 5S, 16S and 23S rRNA and
tRNA
▪ Cleaved by RNases, not by splicing
▪ RNA at both ends of 30S cleaved
▪ Intervening sequences between the different RNAs are removed
▪ Before cleavage 30S RNA precursor are:
▪ methylated at specific bases
▪ Uridine residues are converted to pseudouridine/dihydrouridine
▪ Nucleosides modified in 16S and 23S

▪ E.coli encodes 7 pre-rRNA molecules scattered throughout the

genome
▪ All these genes the identical rRNA coding regions
▪ Sequences in between rRNA regions differ Figure 26.23
 Eukaryote rRNA Processing
▪ A 45S pre-rRNA synthesized by RNA
Polymerase I
▪ 45S pre-rRNA processed in nucleolus to
form 18S, 28S and 5.8S rRNA
▪ 45S pre-rRNA transcript processing
includes:
▪ Cleavage reactions with endo-or
exoribonucleases
▪ Nucleoside modifications
▪ Some pre-rRNAs include introns that must
be spliced Figure 26.24
▪ Small nucleolar RNA’s snoRNAs found in
protein complexes (snoRNPs)
▪ Guide nucleoside modification &
▪ Cleavage
 tRNA Processing
▪ Most cells have 40-50 distinct tRNA’s (20 amino acids,
64 anticodons)
▪ Eukaryotic have multiple copies of many t-RNA genes
▪ Long precursor tRNAs undergo:
▪ Cleavage by endonucleases (RNase P) at 5’ end.
▪ Cleavage by exonucleases (RNase D) at 3’ end.
▪ Addition of CCA at 3’ end by (tRNA nucleotidyltransferase).
▪ Splicing of intron by endonuclease (in eukaryotes).
▪ Modification (editing) of bases
▪ Methylation
▪ Deamination
▪ Reduction
▪ Modified bases occur at characteristic positions in all tRNAs
 tRNA Processing
(Yeast tRNATyr)

Endonuclease tRNA
nucleotidyltransferase
Exonuclease

Endonuclease

Fig 26.26; p1080

 Special function RNA
Processing: snRNA & snoRNA
▪ snRNAs & snoRNA
▪ Facilitate RNA processing
▪ Synthesized as larger precursors and then processed
▪ snoRNAs
▪ Encoded within introns of other genes
▪ As introns spliced from pre-mRNA snoRNP proteins bind to
snoRNA sequences
▪ Ribonucleases remove the extra RNA at 5’ and 3’ ends
▪ snRNAs
▪ snRNA destined for spliceosomes are synthesized as pre-
snRNA by RNA polymerase II
▪ Ribonucleases remove extra RNA at each end
▪ Particular nucleosides in snRNAs modified
 Special function RNA
Processing: miRNA
▪ MicroRNAs (miRNAs)
▪ Involved in gene regulation
▪ Non-coding RNAs (around 22 bp long)
▪ Complementary in sequence to particular regions
of mRNA
▪ Promote mRNA degradation and suppress
translation
▪ Synthesized from much larger precursors in
several steps
 Ribozymes
▪ Cleave themselves or another RNA
▪ 3-D structure integral to function
▪ Inactive if denatured
▪ Show Michaelis-Menton kinetics
▪ Saturable, have active site, have measureable KM, can be
competitively inhibited
▪ Most of activities of ribozymes based on two fundamental
reactions:
▪ Transesterification
▪ Phosphodiester bond hydrolysis (cleavage)
▪ Best characterized ribozymes
▪ Self-splicing group I introns
▪ RNase P
▪ Hammer head ribozyme
 Cellular RNAs are degraded at
different rates
▪ RNA life-time is one means of gene regulation
▪ Concentration of any substance present depends on its
rate of synthesis and degradation
▪ Rate of degradation vary greatly for mRNA for different
eukaryotic genes
▪ Gene product needed briefly
▪ half-life of mRNA minutes or even seconds
▪ Gene product needed constantly
▪ mRNA stable over many generations
▪ Half-lives vary from seconds to hours
▪ Vertebrate cell mRNA half-life ~3 hrs
▪ Bacterial mRNAs half-lives shorter ~1.5 mins
 Cellular RNAs degradation
▪ mRNA degraded via ribonucleases
▪ Hairpin structures in mRNA can extend half-life in Bacteria
▪ 3’ poly(A) tail and 5’ cap important in stability of mRNAs in
Eukaryotes
▪ Degradation in bacteria:
▪ Starts with one or several cuts by an endoribonuclease
▪ Followed by 3’→5’ degradation with exoribonuclease
▪ Degradation in eukaryotes:
▪ Shortening the poly(A)tail
▪ Decapping the 5’end
▪ Degrading mRNA in 5’→3’ direction
▪ 3’→5’ degradative pathway (higher eukaryotes)
▪ 3’→5’ exoribonuclease (exosome) involve in processing of 3’
end of rRNA, tRNA and special function RNA also play a role in
degradation of mRNA
 RNA-Dependent Synthesis
of Nucleic Acids
▪ Most DNA and RNA is synthesized from a DNA template;
however, viruses do not fit with the norm.
▪ Certain RNA viruses carry a RNA dependent DNA
polymerase in the virus particle
▪ Reverse transcriptase
▪ During an infection with ssRNA virus containing reverse
transcriptase:
▪ Virus enter host cell
▪ 1st step in infection is to copy viral RNA into DNA
▪ Next step is to degrade RNA strand from the viral RNA-DNA
hybrid and replace with DNA
▪ Resulting duplex DNA can then be incorporated into host DNA
▪ The integrated viral genes can be activated and transcribed
▪ The gene products, viral proteins and viral RNA genome are
packaged as new viruses
 Reverse Transcriptase can
make DNA from RNA
Reverse Transcriptase catalyze three reactions
1. RNA-dependent DNA synthesis
2. RNA degradation
3. DNA-dependent DNA synthesis

▪ contain Zn2+, like DNA Pol

▪ use a primer of tRNA
▪ lack 3’  5’-proofreading, like RNA Pol
 Retroviruses
▪ RNA viruses that contain reverse transcriptase are
known as retroviruses
▪ Retroviruses cause cancer and AIDS
▪ Reverse transcriptase DOES NOT have a 3'-5'
proofreading
▪ Very high error rate typical for RNA viruses
▪ Higher mutation rate & faster viral evolution
▪ Any 2 viral copies are different
▪ Makes hard to make a vaccine because coat protein is always
changing
▪ Reverse transcriptases important reagents to study:
▪ DNA-RNA relationships
▪ DNA cloning techniques
▪ Synthesis of DNA complementary to an mRNA template
▪ DNA prepared in this manner called complementary DNA (cDNA)
 Pharmaceutical Targets for
HIV (Antiretroviral Drugs)

▪ Reverse transcriptase inhibitors

▪ nucleotide or nucleoside analogs

▪ Protease inhibitors
▪ since proteases are used in cleaving proteins for
packaging into new viral particles
 Telomerase

▪ Telomere is a specialized structure at the ends of

linear eukaryotic chromosomes
▪ Consist of many tandem repeats usually of T1-4G1-4
▪ With A-C on the opposing strand
▪ TG strand is longer than its complement, leaves a 3’-
overhang of several hundred bases
▪ Ends of DNA NOT easily replicated using DNA
polymerases
▪ Beyond an end, there is no template for an RNA primer.
▪ Chromosomes are shortened with each generation.
▪ Telomerase adds telomeric sequences to solve this problem
 Mechanism of Telomerase
▪ Telomerase has RNA with CyAx repeat to serve as
template for synthesis of the TxGy strand of the
telomere.
▪ Telomerase binds to the 3’-end of the chromosome and
hangs off so that the RNA template extends beyond it.
▪ Telomere synthesis requires the 3’-end, of a
chromosome as primer and proceeds in the usual
5’ →3’ direction
▪ The gap on the bottom strand is filled in by DNA
polymerases.

▪ Telomeres in human somatic cells gradually shorten as individual

ages
▪ In vitro if telomerase activity is restored cellular lifespan increases markedly.