RNA Metabolism

RNA:

Storage of information
Transmission of information
Catalysis

Types:
mRNA
tRNA
rRNA

DNA to RNA; Transcription

Transcription : Tightly regulated
Transcriptome
DNA dependent RNA synthesis
RNA syntheis; Transcription

Similarities with replication

Polarity
Template.
Initiation, elongation, and termination phases

Differences
Replication requires a primer
Only one DNA strand serves as a template for a particular
RNA molecule in transcription.
DNA-dependent RNA polymerase

1) Requires DNA template, all four ribonucleoside 5'-triphosphates (ATP, GTP, UTP,
and CTP) and Mg2+.

2) Elongates RNA strand by adding ribonucleotide units to the 3'-hydroxyl end, in the
5'-3' direction.

3) The 3'-hydroxyl group acts as a nucleophile, attacking phosphate of the incoming

ribonucleoside triphosphate

(NMP)n + NTP ------+ (NMP)n+1 + PPi

RNA Lengthened RNA

4) The template DNA strand is copied in the 3'-5‘ direction (antiparallel to the new
RNA strand)
5) Each nucleotide in the newly formed RNA is selected by Watson-Crick base-pairing
Initiation
i) Unlike DNA polymerase, RNA polymerase does not require a primer to initiate synthesis.
ii) RNA polymerase binds at specific DNA sequences called as promoters.

iii) The DNA duplex must unwind over a short distance, forming a transcription "bubble."
iv) E. coli RNA polymerase generally keeps about 17 bp unwound that contains 8 bp RNA-
DNA hybrid
Coding strand As the DNA is rewound, the RNA-
DNA hybrid is displaced and the
RNA strand extruded.

The RNA polymerase is in close

contact with the DNA ahead of the
transcription bubble, as well as with
the separated DNA strands and the
RNA within and immediately behind
the bubble.

A channel in the protein funnels new

nucleoside triphosphates (NTPs) to
the polymerase active site.
In E.Coli, a large, complex enzyme with
flve core subunits (α2ββ’w
M. 390,000) and a sixth subunit σ that
binds transiently to the core and
directs the holoenzyme to specific
binding sites on the DNA.

The RNA polymerase holoenzyme of E.

coli exists in several forms, depending on
the type of σ subunit. The most common
subunit is σ 70 with mol.wt of 70,000
Which sets of genes are expressed is determined by the availability of the various σ subunits,
which in turn is determined by several factors: regulated rates of synthesis and degradation, postsynthetic
modifications that switch individual σ subunits between active and inactive forms, and a specialized class
of anti- σ proteins, each type binding to and sequestering a particular σ subunit making it unavailable for
transcription
 The reaction involves

Two Mg2+ ions, coordinated to

the phosphate groups of the
incoming NTP

Three Asp residues (Asp460,

Asp462, and Asp464 in the β′
subunit of the E. coli RNA
polymerase), which are highly
conserved in the RNA
polymerases of all species.

One Mg2+ ion facilitates attack

by the 3′-hydroxyl group on
the α phosphate of the NTP;
the other Mg2+ ion facilitates
displacement of the
pyrophosphate;

The 3'-hydroxyl group acts as a nucleophile, attacking phosphate

of the incoming ribonucleoside triphosphate

(NMP)n + NTP ------+ (NMP)n+1 + PPi

RNA Lengthened RNA
 Promoters
RNA polymerase binds to specific sequences in the DNA called promoters, which direct the
transcription Of adjacent segments of DNA (genes).

By convention, the DNA base pairs that correspond to the beginning of an RNA molecule are given
positive numbers, and those preceding the RNA start site are given negative numbers.

In E coli,, RNA polymerase binding occurs within a region stretching from about 70 bp before the
transcription start site to about 30 bp beyond it. The promoter region thus extends between positions -
70 and +30.

Similarities in two short sequences centered about positions -10 and -35, interaction sites for the σ70
subunit

The sequences are not identical for all bacterial promoters but certain nucleotides that are particularly
common at each position form a consensus sequence

The consensus sequence at the -10 region is [5')TATAAT(3'); the consensus sequence at the -35 region
is (5')TTGACA(3').

A third AT-rich recognition element, called the UP (upstream promoter) element, occurs between
positions -40 and -60 in the promoters of certain highly expressed genes.
The UP element is bound by the α subunit of RNA polymerase.

The efflciency of RNA polymerase is determined in large measure by these sequences, the spacing
between them, and their distance from the transcription start site.
Promoters
Elongation :
i) Elongation (E coli),proceeds at a rate of 50 to 90 nucleotides/s.
ii) Because DNA is a helix, movement of a transcription bubble
requires considerable strand rotation
iii) DNA strand rotation is restricted by DNA-binding proteins
and other structural barriers.
iv) Moving RNA polymerase generates waves of positive
supercoils ahead of the transcription bubble and negative
supercoils behind.
v) In the cell, the topological problems taken care by
topoisomerases
Movement of an RNA polymerase along
DNA tends to create positive supercoils
(overwound DNA) ahead of the
transcription bubble and negative
supercoils (underwound DNA) behind it.
Transcription initiation and elongation
Binding and initiation :

i) The initial interaction of the RNA polymerase with

the promoter leads to formation of a closed complex
(Binding), in which the promoter DNA is stably bound
but not unwound.
ii) A 12 to 15 bp region of DNA—from within the –10
region to position +2 or +3—is then unwound to form
an open complex.
iii) transcription is initiated within the complex, leading to
a conformational change that converts the complex to the
elongation form
iv) movement of the transcription complex away from the
promoter (promoter clearance)
v) Once elongation commences, the σ subunit is released
(can now bind to the enzyme to initiate transcription, in a
cycle sometimes called the σ cycle) and the polymerase
leaves the promoter and become committed to elongation
of the RNA
 The protein NusA (M, 54,430)

i) σ subunit dissociates stochastically and is replaced by NusA

ii) At terminator sequence, RNA synthesis halts, NusA dissociates from the polymerase, and the RNA
polymerase dissociates from the DNA.
iii) The free polymerase can bind any σ subunit that determines the promoter to which the RNA polymerase
will bind in the next round of synthesis.
Termination of transcription

RNA synthesis is processive, high rate of processivity.

If RNA polymerase is released from a transcript prematurely, synthesis can not be
resumed from the same point, it will start all over again, high processivity required

E coli, has at least two classes of termination signals: one p (rho) and the
other p-independent

RNA polymerase pauses at a variety of DNA sequences, some of which are

terminators

Polymerase bypasses the site and continues on its way, or the complex
undergoes a conformational change (isomerization).
Model for p independent termination
.

At the terminator site, intramolecular pairing

of complementary sequences in the newly
formed RNA transcript may form a hairpin
that disrupts the RNA-DNA hybrid and/or the
interactions between RNA and polymerase,
resulting in isomerization.

The second feature is a highly conserved

string of three A residues in the template
strand that are transcribed into U residues
near the 3' end of the hairpin.

An A=U hybrid region at the 3′ end of the

new transcript is relatively unstable, and the
RNA dissociates completely, leading to
termination and dissociation of the RNA
molecule.
rho-dependent termination :
requires the action of a protein factor called
rho, which has an ATP-dependent helicase
activity that is required for translocation of the
protein along the RNA and transcription
termination.

The p-dependent terminators usually

include a CA-rich sequences in template
called a rut (rho utilization) element.

The rho protein travels along the newly

synthesized RNA, chasing the RNA
polymerase.

The formation of a hairpin loop in the RNA

structure causes the RNA polymerase to pause,
allowing the rho protein to catch up with and
displace the RNA polymerase from the
template.
Proofreading activity of RNA polymerase

RNA polymerase lacks a separate proofreading 3 '-5’, exonuclease active site (as in DNA
polymerases),the error rate for transcription is higher than that for chromosomal DNA replication-
approximately one error for every 104 to 105 Ribonucleotides incorporated into RNA.

Because many copies of an RNA are generally produced from a single gene and all RNAs are
eventually degraded and replaced, a mistake in an RNA molecule is of less consequence to the cell
than a mistake in the permanent information stored in DNA

Many RNA polymerases including bacterial RNA polymerase and the eukaryotic RNA polymerase
II do pause when a mispaired base is added during transcription,as mismatched nucleotides from the
3' end of a transcript are removed by direct reversal of the polymerase reaction (Proofreading???)
 RNA polymerases in eukaryotes
Name Makes a-amanitin
RNA pre-rRNA insensitive
Polymerase I (precursor for
18S,5.8S and 28Sr RNAs)

RNA pre-mRNA very sensitive

Polymerase II some snRNAs

RNA pre-tRNA less sensitive

Polymerase III other small RNAs
some snRNAs
 Subunit structure of eukaryotic RNA
polymerases

• All 3 have multiple subunits (8 to 14 ).

• MW for each polymerase is about 500,000
• Some subunits are common to all 3 RNA polymerases
• All 3 RNA polymerases have subunits that are homologous
to the bacterial b, b’ and a subunits.
 Subunits of yeast RNA Pol II

Approximate subunits per

size (kDa) polymerase role / comment
220(RBP1) 1 related to b'
130(RBP2) 1 related to b
40 (RBP3&11) 2 related to a
35 <1
30 2 common to all 3
27 1 common to all 3
24 <1
20 1 common to all 3
14 2
10 1
 Distinct forms of RNA polymerase used for initiation
and elongation: RNA Pol II

CTD has repeat of (YSPTSPT)26-50.

CTD = C-terminal domain
CTD with heptad YSPTSPT- CTD;27 such repeats in yeast and 52 in human

Important for functioning of Pol II, phosphorylated by TFIIH

CDK9 also important for phospphorylation

Phosphoraylation: At Ser, different status at different stages affecting the interaction of

TFs and EFs
 Common sequences in promoters recognized by
eukaryotic RNA polymerase II

The TATA (Goldberg-Hogness box) box is the major assembly point for the proteins of the pre-initiation
complexes of Pol II.

The DNA is unwound at the initiator sequence (Inr), and the transcription start site is usually within or
very near this sequence.

In the Inr consensus sequence ,N represents any nucleotide; Y, a pyrimidine nucleotide.

Many of the sequences (highly variable) are located within a few hundred base pairs of the TATA box on
the 5′ side; others may be thousands of base pairs away.

Many Pol II promoters lack a TATA box or a consensus Inr element or both.

Additional sequences around the TATA box and downstream of Inr may be recognized by one or more
transcription factors.
Transcription at RNA polymerase II promoters •The sequential assembly of TBP (often with
TFIIA), TFIIB, TFIIF plus Pol II, TFIIE, and
TFIIH results in a closed complex.

•The DNA is unwound at the Inr region by the

helicase activity of TFIIH and perhaps of
TFIIE, creating an open complex.

•The carboxyl-terminal domain (CTD) of the

largest Pol II subunit is phosphorylated by
TFIIH,

•Polymerase then escapes the promoter and

begins transcription.

•During synthesis of the initial 60 to 70

nucleotides of RNA, first TFIIE and then TFIIH
is released, pol II enters elongation phase

•TFIIF remains associated with Pol II

throughout elongation.

•TFIIH important for repair for any damage in

the template strand. Whenever Pol II halts at the
site of any DNA lesion, it interacts with the
lesion and recruit the entire nucleotide-excision
repair complex
Elongation, Termination, and Release

• EFs, important for elongation

• Binding of EFs to polymerase (RNA Pol II) increase its activity

• some bound to the phosphorylated CTD, suppress pausing during transcription

• EFs coordinate interactions between protein complexes involved in the

posttranscriptional processing of mRNAs.

• RNA transcript is completed, transcription is terminated. Pol II is

dephosphorylated and recycled to initiate another transcript
 Inhibitors of DNA transcription.
bind to the minor groove of the double helix. Complex of actinomycin D with DNA

intercalates between two

successive G≡C base pairs in
duplex DNA

also by intercalation in DNA

Rifampicin inhibits bacterial RNA synthesis by binding to the B subunit of bacterial RNA
polymerases, preventing the promoter clearance step of transcription
Actinomycin D and Acridine : Inhibitor of both bacterial and eukaryotic transcription
Alpha amanitin : Only eukaryotic Pol II
Eukaryotic mRNA Processing

5’ cap of mRNA
A residue of 7-methylguanosine
linked to the 5'-terminal residue of
the mRNA through an unusual
5',5'-triphosphate linkage.

Methyl groups (pink) are often

found at the 2′ position of the first
and second(only in vertebrates)
nucleotides.
Generation of the 5’Cap

GTP : for guanosine

S-adenosylmethionine : Methyl gp
donor

All these reactions occur very early in

transcription, after the first 20 to 30
nucleotides of the transcript have
been
added

The 5' cap protect mRNA from

ribonucleases

It binds to specific Cap binding

complex of proteins and participates
in binding of the mRNA to the
ribosome to initiate translation
Synthesis of the cap is carried out by enzymes
complexed to the CTD of Pol II

The cap remains tethered to the CTD through

an association with the cap-binding complex
(CBC).
Both Introns and Exons are transcribed from DNA to RNA

In bacteria: polypeptide chain has amino acid sequence co-linear with

DNA seq.

But not so in case of eukaryotes due to presence of interrupting

sequences in the genes which are non coding : Introns (exception
histones)

Genes of higher eukaryotes, including humans, typically have

much more DNA devoted to introns than to exons

Functional RNA: splicing of introns and joining of exons

Group I and II introns differ in distribution, structure, catalysis method,

ORF domain.

Two other groups of introns, III, IV require Spliceosomal machinery for

Splicing modification
 Group I Group II

• Self splicing
• Self splicing • No enzyme required
• No enzyme required • primarily in
• In : mitochondrial, nuclear chloroplast/mitochondrial
and chloroplast genes genes for mRNA in fungi,
encoding rRNA, mRNA, algae and plants
tRNA • No ATP requirement
• No ATP requirement

Both differ in their splicing

mechanism
Splicing of Group I introns: Transesterification reaction
Requires a guanine nucleoside. or nucleotide cofactor

3’OH of a guanosine (may be GMP,GDP or GTP) molecule acts as nucleophile,

attacking the phosphodiester linkage between U and A residues at an exon-intron
junction of an mRNA molecule
The nucleophile in the first step may be guanosine, GMP, GDP, or
GTP. The spliced intron is eventually degraded.
Splicing mechanism of group II introns

The mechanism is same as that of

Group I except that nucleophile in
the first Step is 2’OH group of an
A residue within the intron

Formation of 2’5’phospodiester bond

instead of routine 3’5’phosphodiester
bond as in case of Group I introns
 Spliceosome mediated splicing
•Third class of introns: spliceosomal introns

•Found in nuclear mRNA primary transcript

•Removal catalysed by spliceosome

•Spliceosome: RNA protein complexes, small nuclear ribonucleoproteins (snRNPsor snurps)

•Snurps have RNAs 100-200nucleotides,small nuclear RNA(snRNAs)

•snRNAs: U1,U2,U4,U5,U6 involved in splicing, present in nuclei

•Mechanism: Lariat formation

•Spliceosome introns: GU at 5’ and AG at 3’; sites for splicing

•U1snRNA has sequence complementary to 5’splice site

•Although spliceosomal introns seem to be limited to eukaryotes , Genes with group I and II
introns have now been found in both bacteria and bacterial viruses
Splicing mechanism for spliceosomal introns: Group III

Spliceosome:

•Spliceosomal introns generally have the dinucleotide sequence GU at the 5' end and AG at the
3' end, and these sequences mark the sites where splicing occurs.

•The U1 snRNA has a sequence near its 5′ end complementary to the splice site at the 5′ end of
the intron followed by addn of U2, U4,U5and U6 along with about 50 proteins to form
spliceosome

•Some times U11 and U12 instead of U1 and U2

Assembly of spliceosomes

Remaining snRNPs (the U4-U6

complex and U5) bind to form
an inactive spliceosome.

ATP required for assembly only

Internal rearrangements

U1 and U4 expelled

U6 is paired with both the 5′

splice site and U2.

Active spliceosome

Catalytic steps similar to group II

introns
Coordinated splicing and transcription

After splicing, introns remain in the nucleus and get degraded

 Fourth class of introns

Present in some tRNAs

Splicing reaction requires ATP and endonuclease

Endonuclease cleaves the phosphodiester bond at the

two ends of the introns

Ligation of exons similar to DNA ligase reaction

Addition of the poly(A) tail to the primary RNA transcript of eukaryotes

At their 3' end, most eukaryotic mRNAs have a string of

80 to 250A residues making up the poly(A) tail.

Poly(A) tail serves as a binding site for one or more specific

proteins.

The poly(A) tail and its associated proteins probably protect

mRNA from enzymatic destruction.

Many bacterial mRNAs also acquire poly(A) tails, but

these tails stimulate decay of mRNA rather than protecting
it from degradation.
Addition of the poly(A) tail to the primary RNA transcript of eukaryotes

Pol II synthesizes RNA beyond the segment of the

transcript containing the cleavage signal sequences
the highly conserved upstream sequence
(5′)AAUAAA.

1 The cleavage signal sequence is bound by an

enzyme complex that includes an endonuclease, a
polyadenylate polymerase, and several other
multisubunit proteins involved in sequence
recognition, stimulation of cleavage, and regulation
of the length of the poly(A) tail.

2 The RNA is cleaved by the endonuclease at a point

10 to 30 nucleotides 3′ to (downstream of) the
sequence AAUAAA.

3 The polyadenylate polymerase synthesizes a

poly(A) tail 80 to 250 nucleotides long, beginning at
the cleavage site.
Thus, Cleavage generates the free 3’-hydroxyl group that
defines the end of the mRNA, to which A residues are
immediately added by polyadenylate polyrmerase, which
catalyzes the reaction

RNA + nATP ------ RNA-(AMP)n + nPPi

where n : 80 to 250. This enzyme does not require a

template but does require the cleaved mRNA as a primer
Overview of the processing of a eukaryotic mRNA
 Multiple products by differential RNA processing

By more than one site for cleavage and polyadenylation or alternative splicing patterns, or both.

Alternative cleavage and polyadenylation patterns in eukaryotes

This mechanism, called poly(A)site choice, generates diversity in the
variable domains of immunoglobulin heavy chains
Alternative splicing patterns
 Processing of pre-rRNA transcripts in bacteria
Before cleavage, the 30S RNA precursor is
methylated at specific bases (red tick marks)
either on bases or on 2′-hydroxyl groups, some
uridine residues are converted to pseudouridine
6500 nucleotides (blue tick marks) or dihydrouridine (black tick
mark) residues.

Cleavage liberates precursors of rRNAs and

tRNA(s).

Cleavage at the points labeled 1, 2, and 3 is

carried out by the enzymes RNase III, RNase P,
and RNase E, respectively. RNase P is a
ribozyme.

The final 16S, 23S, and 5S rRNA products result

from the action of a variety of specific nucleases.

The seven copies of the gene for pre-rRNA in the

E. coli chromosome differ in the number,
location, and identity of tRNAs included in the
primary transcript.

Some copies of the gene have additional tRNA

gene segments
Some modified bases of rRNAs and tRNAs, produced in
posttranscriptional reactions

96 modified nucleosides known to occur in different RNA species

81 different types known in tRNAs
30 in rRNAs.
 Processing of pre-rRNA transcripts in vertebrates
• 45S pre-rRNA transcript is synthesized by RNA
polymerase I in nucleolus, incorporated into a
nucleolar 90S preribosomal complex

• Tight coupling between rRNA transcription,

rRNA maturation and ribosome assembly

• The 45S precursor is methylated at more than

100 of its 14,000 nucleotides, either on the
bases or on the 2′-OH groups

• Some uridines are converted to pseudouridine,

and a few other modifications occur.

• A series of enzymatic cleavages of the 45S

precursor produces the 18S, 5.8S, and 28S
rRNAs, and the ribosomal subunits gradually
take shape with the assembling ribosomal
proteins.
Synthesized separately by Pol III
Small nucleolar RNAs (snoRNAs)
Guide nucleoside modification and Cleavage reactions
60 to 300 nucleotides with 10 to 21 nucleotides perfectly complementary to
some site on an rRNA.

Found in protein complexes (snoRNPs) in the nucleolus , bound to 4-5

proteins, include the erzymes that carry out the modification

Two classes of snoRNPs , defined by key conserved sequence elements

referred to as lettered boxes.

The box H/ACA snoRNPs - involved in pseudouridylylation

The box C/D snoRNPs function in 2'-O methylations

Unlike bacteria , same enzyme may participate in modifications at many sites

guided by snoRNAs.

Conserved sequences fold into structures that are bound by the snoRNP
proteins
 The function of snoRNAs in guiding rRNA modification
(a)RNA pairing with box C/D snoRNAs to guide methylation reactions. The methylation sites in
the target rRNA (dark green) are in the regions paired with the C/D snoRNA. The highly
conserved C and D (and C′ and D′) box sequences are binding sites for proteins that make
up the larger snoRNP.
(b)RNA pairing with box H/ACA snoRNAs to guide pseudouridylylations. The pseudouridine
conversion sites in the target rRNA (green segments) are again in the regions paired with
the snoRNA, and the conserved H/ACA box sequences are protein-binding sites.

Methylation
sites

Pairing with
Boxes guiding
methylation
 Processing of tRNAs in bacteria and eukaryotes

Longer RNA precursors: enzymatic removal of nucleotides from the 5' (The endonuclease RnaseP )and
3' ends (exonuclease RNase D) : tRNA

In eukaryotes, introns are present in a few tRNA transcripts(removed at the final step of processing)

3'-terminal trinucleotide CCA(3') added by tRNA nucleotidyltransferase, an unusual enzyme that binds
the three ribonucleoside triphosphate precursors in separate active sites

Final modiflcation of some bases by methylation, deamination, or reduction .

In the case of pseudouridine, the base (uracil) is removed and reattached to the sugar through C-5.
 Micro RNAs(miRNAs)

• A special class of RNAs involved in gene regulation

• Non-coding

• ~22 nucleotides long

• Complementary in sequence to particular regions of mRNAs

• Regulate mRNA function by cleaving the mRNA or suppressing

its translation.

• Up to 1% of the human genome may encode miRNAs

Synthesis and processing of miRNAs
Primary transcript : Large pri-miRNA.

Processing : by two endoribonucleases (RNase III

family), Drosha and Dicer.

In the nucleus, the primiRNA is reduced to a 70 to 80

nucleotide precursor miRNA (premiRNA) by a
protein complex including Drosha and another
protein, DGCR8.

The pre-miRNA : exported to the cytoplasm, acted

on by Dicer , produce the nearly mature miRNA
paired with a short RNA complement.

RNA complement removed by an RNA helicase,

mature miRNA is incorporated into protein
complexes, RNA-induced silencing complex (RISC),
which then bind a target mRNA.

If the complementarity between miRNA and its

target is nearly perfect, the target mRNA is cleaved.
If the complementarity is only partial, the complex
blocks translation of the target mRNA.
 RNA Enzymes: Ribozymes

The best-characterized ribozymes are the self-splicing group I introns,

RNase P, and the hammerhead ribozyme

The activity of ribozymes based on two reactions:

Transesterification
Phosphodiester bond hydrolysis

Substrate is often a RNA molecule, may be part of ribozyme itself

Base pairing interactions to align the substrate RNA

Hammerhead ribozyme: shaped like the head of a hammer
The boxed nucleotides are highly conserved and are required for
catalytic function. The arrow indicates the site of self-cleavage.

The boxed nucleotides are highly conserved and are required for
catalytic function. The arrow indicates the site of self-cleavage.
Enzymatic Properties of Group I Introns as Ribozymes
• Exhibit catalytic activity like enzymes
• Intron sequences are shaded yellow,
exon sequences green.
• Each thick yellow line represents a bond
between neighboring nucleotides in a
continuous sequence
• The catalytic core of the self-splicing
activity is shaded. The P1 region, which
contains the internal guide sequence
(boxed), is the location of the 5′ splice
site (red arrow).
• Part of the internal guide sequence pairs
with the end of the 3′ exon, bringing the
5′ and 3′ splice sites (red and blue
arrows) into close proximity.. This
pairing promotes the alignment of
specific bonds to be cleaved and
rejoined
 Polynucleotide Phosphorylase : Bacterial Enzyme

• (NMP)n + NDP ============= (NMP)n+1 + Pi

• Uses the 5'-diphosphates of ribonucleosides (with any or all of the four) as

substrates , cannot use homologous 5'-triphosphates and deoxyribonucleoside 5'-
diphosphates

• Not template dependent, polymer it forms does not have a specific base sequence

• The base composition reflects relative concentrations of the 5'-diphosphate in the

medium

• RNA synthesized contains the usual 3',5'-phosphodiester linkages, hydrolyzed by

ribonuclease

• Reversible reaction, can be pushed in the direction of breakdown of the

polyribonucleotide by increasing the phosphate concentration.

• The probable function is the degradation of mRNAs to nucleoside diphosphates

 RNA-Dependent Synthesis of Nucleic Acids
• Most DNA and RNA is synthesized from a
DNA template; however, viruses do not fit
with the norm.
• Retroviruses have genomes of ssRNA and
the enzyme reverse transcriptase.
– virus enters host cell
– reverse transcriptase makes DNA from
the RNA
• It then degrades the RNA from the
DNA-RNA hybrid and replaces it with
DNA.
• DNA can then be incorporated into
host DNA.
Retroviral Infection of a Mammalian Cell and
Integration into Host Chromosome

• Viral particles entering the host cell

carry viral reverse transcriptase and a
cellular tRNA (picked up from a former
host cell) already base-paired to the
viral RNA

• The tRNA facilitates immediate

conversion of viral RNA to double-
stranded DNA by the action of reverse
transcriptase

• Double-stranded DNA enters the

nucleus and is integrated into the host
genome catalyzed by a virally encoded
integrase
 Retroviruses Typically Contain Three
Genes Plus a Long Terminal Repeat

• gag (group associated antigen)

– encodes a long polypeptide that is cleaved into six
smaller proteins that make up viral core
• Pol (polypeptide)
– encodes protease that cleaves the long polypeptide,
reverse transcriptase, and an integrase to insert DNA
into host genome
• env
– encodes viral envelope
• Long terminal repeat (LTR) facilitates integration
of virus genome into host DNA
 Structure and Gene
Products of an
Integrated Retroviral
Genome
 Reverse Transcriptases 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
- make reverse transcriptase error-prone
- explains high rate of virus mutation/evolution (1: 20,000
nt)
 Some Retroviruses Cause Cancer

• Some retroviruses contain an oncogene.

– Example: The Rous sarcoma virus has the
src gene.
• Src for sarcoma, a cancer of bone, fat, muscle,
etc. (vs. cancer of epithelial cell origin)
• encodes a non-receptor tyrosine kinase, an
enzyme that affects cell division
 HIV Retrovirus Causes AIDS

• The HIV genome has genes for killing the host (mostly T
lymphocytes).
– results in suppression of immune system
• HIV-encoded reverse transcriptase is unusually error
prone.
– complicates push for vaccine
– at least one error per replication, so potentially no two viral RNAs
alike
 Pharmaceutical Targets for HIV (Antiretroviral Drugs)

• Reverse transcriptase inhibitors

– nucleotide or nucleoside analogs
– drug names ending in “dine” or “sine”:
• zidovudine (AZT), didanosine (Videx), and so on
• Protease inhibitors
– since proteases are used in cleaving proteins
for packaging into new viral particles
– drug names ending in “avir”:
• indinavir, saquinavir, and so on
 Retrotransposons in Eukaryotes
Have Similarities to Retroviruses
• Retrotransposons are mobile genetic elements in
eukaryotes.
– encode an enzyme with homology to reverse
transcriptase of retroviruses
– move between positions via RNA
intermediates
• using their enzyme to make DNA from RNA
– unlike bacterial transposons that move
directly from DNA to DNA
 Retrotransposons

• Lack the env gene, so don’t form viral particles

• Examples: Ty element in yeast and Copia element in
Drosophila
 Telomeres

• Structures at the ends of linear eukaryotic chromosomes

• Have tandem repeats usually of T1-4G1-4
- with A-C on the opposing strand
• Can be tens of thousands of bp long in mammals
• TG strand is longer than its complement, leaves a 3’-
overhang of several hundred bases
 Telomerase Extends the Ends of Chromosomes

• Telomeres are 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.
 The 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.
• Telomerase extends the 3’-end, using the RNA
of the enzyme as the primer.
• The gap on the bottom strand is filled in by DNA
polymerases.
Telomere Synthesis and Structure
The internal template RNA of
telomerase binds to and basepairs with
the TG primer (TxGy) of DNA.

Telomerase adds more T and G residues

to the TG primer, then repositions the
internal template RNA to allow the
addition of more T and G residues that
generate the TG strand of the telomere.

The complementary strand is synthesized

by cellular DNA polymerases after
priming by an RNA primase.

The single-stranded tail synthesized by

telomerase is folded back and paired with
its complement in the duplex portion of
the telomere.

The telomere is bound by several

telomere-binding proteins, including
TRF1 and TRF2 (telomere repeat
binding factors).