Lec #1 RNA Synthesis and Processing

Information stored in stable form in DNA is available for

 replication for the continuation of generations
 transcription (DNA→RNA) for metabolism
 RNA transcripts in eukaryotes are extensively modified, capping of
the 5’ end of an mRNA precursor and the addition of a long
poly(A) tail to its 3’ end.
 One of the most striking examples of RNA modification is the
splicing of mRNA.
 In addition, individual bases in some pre-mRNA molecules are
changed in a process called RNA editing.
Stages in Transcription

Transcription involves
 Initiation: involving promoters and other elements
 Elongation: interaction of RNA Pol, DNA template & nascent
RNA chain
 Termination: Dissociation of RNA pol from template
 Processing: Post-transcriptional changes in RNA

An overview of RNA synthesis

• RNA synthesis or transcription is a process of transcribing the DNA
sequence information into RNA sequence information
• Catalyzed by RNA polymerases
• Basic biochemistry of RNA synthesis is common in pro- and eukaryotes
• Similarity is also evident from 3D structure of respective RNA
polymerases
• Polymerase structure is similar, but not identical indicating regulation is
more complex in eukaryotes

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Actions of RNA polymerase
RNA pol. searches DNA sequences (promoters) for initiation
 Promoters are also called cis-acting elements
 Unwinds a short stretch of double-helical DNA to produce single stranded
DNA template,
 Uses only one strand as template i.e. the anti-sense, non-sense or non-
coding strand
 Selects the correct NTP for polymerization
 RNA pol. searches DNA sequences (promoters) for initiation
 Promoters are also called cis-acting elements
 Unwinds a short stretch of double-helical DNA to produce single stranded
DNA template,
 Uses only one strand as template i.e. the anti-sense, non-sense or non-
coding strand
 Selects the correct NTP for polymerization
Bond formation in DNA &RNA
• The fundamental process of phospho-diester bond formation is same as
that by DNA polymerases
• Three aspartate residues in RNA Pol active site take part in reaction, and
2 Mg2+ are also involved
• One Mg2+ is enzyme bound, while the other comes in with NTP and
leaves with pyrophosphate
RNAs: Similarities & Differences
• Basic biochemistry is same for mRNA, rRNA and tRNA, but they all
RNAs differ in their
• size
• Regulation
• posttranscriptional modifications and
• in eukaryotes, involvement of specific RNA polymerase

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 RNA polymerase Composition
• E. coli RNA polymerase is a large 400 kDa complex enzyme
• Consists of four kinds of subunits
• Holoenzyme is α2ββ’σ, products of genes rpoA, rpoB, rpoC and rpoD
(σ70) respectively
• The σ subunit helps to find promoter site
• The RNA polymerase without σ unit is termed as core enzyme (α2ββ’ )
• Catalytic site for polymerization is located in core enzyme

INITIATION
• Transcription is initiated at promoter sites on the DNA template
• Promoters are sequences of DNA that direct the RNA polymerase to the
proper initiation site for transcription.
• Promoters can be characterized by a technique called "foot-printing”
Promoters
• In prokaryotes two common motifs are present on the 5’ side of the start
site
• These sequences each are 6 bp long and centered at about -10nt
(TATAAT) and -35nt (TTGACA) upstream of the transcriptional start site
• Promoters differ markedly in their efficiency
• The consensus (average) sequence from promoter can be determined
Consensus elements

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  Strong promoters in E. coli. transcribe their genes after every two
seconds
 Weak promoters may transcribe as less as once in ten minutes
 Single mutation in promoter sequence or even distance between these
sequences can diminish the activity of promoter
 The efficiency or strength of a promoter sequence serves to regulate
transcription.
Promoter recognition by RNA Pol
 Sigma subunit of RNA polymerase recognize promoter sites
 The core RNA polymerase is unable to start transcription at promoter site,
rather holoenzyme is required
 The σ functions in specific initiation in two ways:
 It decreases the affinity of RNA polymerase for general regions of DNA
by a factor of 104
 The holoenzyme binds to duplex DNA with the help of σ factor and
moves along the DNA in search of a promoter
 The search is rapid and unidirectional rather than 3-D
 So it is 100 faster than as it could be by repeated binding and releasing
 σ recognize promoter by α-helix on its surface
 Multiple σ factors in E. coli. recognize several type of promoters

Un-winding Template
 Transcription requires unwinding of the template double helix
 After finding the promoter, for transcription to start, RNA polymerase
must unwind duplex DNA to use one strand as template
 RNA polymerase has the catalytic activity to unwind a 17 bp segment of
DNA, which is about 1.6 turns of B-DNA helix

Transcription Bubble
RNA chains are formed de novo without the need of a primer
 RNA chain like DNA, always grow in the 5’ to 3’ direction

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  Nascent RNA chains carry a highly distinctive tag on the 5’ end : the first
base is either pppG or pppA
 Initiation is completed by the time first phosphodiester bond is formed

Elongation
 Elongation starts from the addition of 3rd nucleotide to the RNA chain
(formation of second phosphodiester bond)
 σ subunit dissociates after addition of 9-10nts, while core enzyme binds
with higher strength to DNA template
 Rest of the elongation is performed only by core enzyme
 Elongation takes place in transcription bubbles that moves along the DNA
template
 Elongation bubble comprises of RNA pol, DNA template and nascent
RNA
 Nascent RNA forms hybrid with DNA which is ~8 bp long
 The hybrid is continually dissociated
 3’-OH of nascent chain is positioned to attack α-P of incoming NTP
 Core RNA Pol also keeps hold of other strand (coding strand) by a
separate site
 The rate of elongation is about 50 nt.s-1 compared to the DNA synthesis
which is about 1000 nt-1 (on one strand)

Fidelity of Transcription
Termination
 Termination may be
 Protein Independent
 Protein Dependent
Protein Independent Termination
 An RNA hairpin followed by several uracil residues terminates the
transcription of some genes

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  As in case of initiation of transcription, termination is also precisely
controlled
 The transcribed regions of DNA templates contain stop signals
 The simplest one is a palindromic GC
-rich region making a self
complimentary hairpin loop followed
by an AT-rich region
o RNA–DNA hybrid helix produced after the hairpin is unstable because its
rU–dA base pairs are the weakest of the four kinds.
o Hence, the pause in transcription caused by the hairpin permits the
weakly bound nascent RNA to dissociate from the DNA template and
then from the enzyme.

Protein Dependent termination

Rho Dependent Termination
o The Rho protein helps terminate the transcription of some genes
o Addition of Rho at different times after initiation yields RNA molecules
of different sizes
o There may be more than one binding sites for Rho on a single transcript

 A hexameric Rho complex binds ~72 nts of RNA depending on

high G:C ratio
 Rho degrades ATP only in the presence of single stranded RNA
 Then slides towards transcription bubble

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  Acting as RNA:DNA hybrid Helicase, Rho breaks the
RNA:DNA hybrid

Other Termination Proteins

 Other mechanism for transcriptional termination do exist i.e.
 nusA protein enables RNA polymerase in E. coli to recognize a
characteristic class of termination sites.
 attenuation, a mechanism for transcriptional control of amino acids
synthesis, etc.
 A common feature of protein-dependent and protein-independent
termination is that the functional signals lie in newly synthesized RNA
rather than in the DNA template

Some messenger RNAs directly sense

metabolite concentrations
 Some mRNA molecules form special secondary structures that directly
binding small molecules -termed riboswitches.
 Flavin mononucleotide (FMN)-key intermediate in riboflavin
biosynthesis in Bacillus subtilis.
 FMN when present at high concentration, it binds to a conformation of
the RNA transcript favoring hairpin structure that favors premature
termination.

RNA Modifications in Prokaryotes

mRNA
• mRNA in prokaryotes undergoes no regular/significant
modification after transcription
• Translation starts while transcription is still going on

rRNA Modifications
• Precursors of ribosomal RNA are cleaved and modified after
transcription

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 • In E. coli all three types of rRNA molecules and a tRNA molecule
are excised from a single primary RNA transcript that also contains
spacer region
• Ribonuclease III excise 5S, 16S and 23S rRNA precursors from
primary transcript by cleaving double helical hairpin regions at
specific sites
• Other changes include enzymatic modification of nucleotides e.g.
methylation of adenosine to 6-dimethyladenosine

tRNA Modifications
• Precursors of transfer RNA are also cleaved and enzymetically
modified after their transcription
• pre-tRNAs are synthesized in the form of arrays of several kind of
tRNAs or several copies of the same tRNA
• Ribonuclease P (a ribonucleoprotein containing catalytic RNA)
generates correct 5’-termini of all tRNA molecules
• Ribonuclease D finish 3’-end of tRNAs

Other Modifications in tRNA

• Addition of nucleotides to the termini of some tRNA chains. e.g.
CCA to some tRNA 3’ ends, if not already present
• CCA act as amino acid binding site on tRNA
• Unusual bases are also found in all types of tRNAs e.g. Uracil
residues are enzymetically converted into ribothymidine and
pseudouridine
• The modifications generate diversity which allow greater structural
and functional diversity

Inhibitors of transcription
Rifampicin
• Rifampicin in an antibiotic

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 • It is semi-synthetic derivative of rifamycin, derived from a strain of
Streptomyces
• It inhibits the initiation of RNA synthesis by interfering with the
formation of first few phosphodiester bonds.
• It blocks the channel into which the RNA-DNA hybrid is generated
by the enzyme
• It does not hinder chain elongation once initiated

Actinomycin D
• It is a polypeptide containing antibiotic activity, obtained from a
different strain of Streptomyces
• It blocks RNA synthesis by binding tightly and specifically to
double-helical DNA by intercalation and thereby prevents it from
being an effective template for RNA synthesis.
• It does not bind with ssDNA, ssRNA , ds RNA or RNA-DNA
hybrids
• At higher concentrations it may inhibit replication.
• These properties make it highly specific inhibitor of the formation
of new RNA in both prokaryotes and eukaryotes.
• Due to its ability to inhibit rapidly growing cells, it is an effective
agent in treatment of some cancers

Lec #2: RNA Synthesis in Eukaryotes

Transcription in Eukaryotes
 Much more complex phenomenon than in prokaryotes
 Eukaryotic transcription and translation are separated in space and time
 This separation has enabled eukaryotes to evolve post-transcriptional
regulatory strategies
 Transcription is directed by
 a single type of RNA polymerase in prokaryotes
 while four types of RNA polymerases are known in eukaryotes

Eukaryotic RNA Polymerases

 In eukaryotes there are at least four types of RNA polymerase,
which differ in
 template specificity

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  location within nucleus
 Susceptibility to inhibitors

 All these polymerases are large proteins, containing from 8 to 14

subunits and having a total molecular mass greater than 500 kDa
 These RNA polymerase are homologous to each other and to
prokaryotic RNA polymerase, but RNA polymerase II contains a
unique carboxyl-terminal domain
 Activities of RNA Pol II are regulated by serine/ threonine
phosphorylation present in carboxyl-terminal domain

spRNAP-IV
 spRNAP-IV i.e. single-polypeptide RNA polymerase-IV was recently
reported
http://www.nature.com/nature/journal/v436/n7051/abs/nature03848.html
 It is a homolog of mitochondrial RNA polymerase
 Localized in the nucleus
 Transcribes some genes in humans and rodents
 It is insensitive to α-amanitin but is sensitive to short interfering RNA
specific for the POLRMT gene (coding for mtRNA Pol)
 The promoters for spRNAP-IV differ substantially from those used by
RNA polymerase II
 It does not respond to transcriptional enhancers
α-Amanitin Sensitivity
 α-Amanitin is produced by a poisonous mushroom Amanita phalloides
(commonly called death cup or destroying angel)
 It is a cyclic octapeptide that contains several modified amino acids
 It binds very tightly (kd= 10nM) to RNA polymerase II and blocks the
elongation phase of transcription
 It also inhibit RNA polymerase III but at much higher concentrations
(1uM)
 RNA polymerase I is insensitive to this toxin
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 Transcriptional Elements
1. Cis-acting elements
i. Promoters
ii. Enhancers
2. Trans-acting elements
Cis-acting Elements
Promoters
 Eukaryotic RNA Polymerases also need specific promoter for
transcriptional initiation
 Eukaryotic promoters also consist of conserved regions
 Promoters for different RNA Polymerases differ in sequence and position
 Promoters are
 generally located
○ upstream of the gene, however, they may be
○ embedded either in the gene or may be
○ located downstream
Promoters of RNA Pol-I
 It transcribes ribosomal DNA (rDNA?)
 Genes are arranged in several hundred tandem repeats
 Each repeat containing one copy each of 28S, 18S and 5.8S rRNA genes
 Promoters elements are situated in stretches of DNA separating the genes
 TATA-like sequence called ribosomal initiator element (rInr) is found at
transcriptional start site
 An Upstream Promoter Element (UPE) is also available between -150 to
-200
 Both these elements bind to proteins that help to recruit RNA
Polymerase-I

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Promoters of RNA Pol-II

 Promoter of RNA Pol-II are located on 5’ side of the start of transcription

 For RNA Pol-II TATA box is the most important cis-acting element which
is situated between position -30 and -100
 TATA box may be compulsory but not sufficient
 Promoter can contain any combination of a number of possible elements
 May also involve enhancer element, unique to eukaryotes

Promoters of RNA Pol-III

 Located within transcribed sequence, downstream of the start site
 These are of two types
 Type I found in 5S rRNA genes containing two short conserved
sequences called A block and C block
 Type II found in tRNA genes consists of two 11bp sequences called
A block and B block, situated 15 bp from either end of the gene

Enhancers
 Eukaryotes and their viruses contain DNA sequences called enhancers
 These are not promoters by themselves but enormously increase
effectiveness of the promoters
 Position of the enhancers is not fixed and vary greatly
 Enhancer sequences can stimulate transcription at start sites located
thousands of nucleotides away

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  Enhancers may be located upstream, downstream or even in the midst
of a transcribed gene
 They influence the gene regulation in specific tissues and at specific
developmental stages e.g.
 the immunoglobulin enhancer functions in B lymphocytes but not
elsewhere
 In Burkit lymphoma and B-cell leukemia, a chromosomal translocation
brings the proto-oncogene myc (a transcriptional factor itself) under the
control of a powerful immunoglobulin enhancer, which results in
progression of cancer
 Presence of similar promoters and influence of similar enhancer create
batteries of genes operative in a given set of internal/external
environmental conditions

Trans-acting elements/factors
 Cis-acting elements in eukaryotes are binding sites for proteins called
Transcription Factors
 These proteins are regarded as trans-acting elements/ factors because
their genes may be located on a different DNA molecules (other than
their target gene)
 These proteins generally called Transcription Factor help RNA
polymerase to bind to its promoter

Transcription Complex
 Transcription Factor of RNA Pol-II are called TFII
 Individual TFII are named as TFIIA, TFIIB and so on....
 The TATA Box-Binding Protein (TBP), a component of TFIID, initiates
the assembly of the active transcription complex
 TBP binds at TATA box 105 more tightly than non TATA box sequence.
 The binding of TBP brings the conformational changes in DNA and
TBP
 Surface of the bound TBP serves as docking site for binding of other
proteins
 This is followed by sequential joining of TFIIA and TFIIB
 Then the complex is joined by TFIIF, an ATP driven Helicase, which is
responsible for initial unwinding, for action of RNA Pol II to start.
 Finally joining by RNA Pol II, TFIIE and TFIIH result in formation of
complex called “basal transcription apparatus”

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  Phosphorylation of the C-terminal domain of RNA Pol II (CTD) at
serine and threonine residues by TFIIH, marks the transition from
initiation to elongation.

Evolution of Euk. Transcription

 Bacteria lack TBP, however archaea do have TBP
 Archaea have three different RNA polymerases
 Generally archaea transcriptional control process resemble more to
eukaryotes than to bacteria
 Thus many eukaryotic transcriptional components seem to have been
evolved from an ancestor of archaea
 Multiple transcriptional factors interact with eukaryotic promotes
 The basal transcriptional assembly initiate transcription with relatively
low frequency
 Many other transcriptional factors join the assembly to
 modulate rate
 selectively stimulate specific genes e.g.
 CTF (CAAT Transcription Factor) also called NF1 binds CAAT box
 Sp1 binds to promoter containing GC box (a mammalian protein ~100
kd)
 SV40 (a virus producing caner in monkeys) duplex DNA contain five
GC box in -50 to -100 region
 HSTF (Heat Shock Transcription Factor) in Drosophila bind to a
HSRE located 15 bp upstream of TATA box (CNNGAANNTCCNNG)

Lec#3 Processing of Eukaryotic RNA

▪ Processing of tRNA
➢ All the three types of eukaryotic transcriptional products are processed
➢ tRNA precursors are converted into mature tRNAs by a series of
alterations
➢ Cleavage of a 5’ leader sequence,
➢ Splicing to remove an intron,
➢ Replacement of the 3’ terminal UU by CCA
➢ Modification of several bases

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 ➢ A series of enzymes act on RNA chain or its constituent bases to achieve
the final product

Processing of rRNA
RNA Pol-I makes a single precursor rRNA of (45S in mammals)
 First the precursor undergo extensive modifications on
 Bases
 Ribose
 These changes are directed by small nucleolar ribonuclear proteins
(snoRNPs)
 Each snoRNP contains one snoRNA and many proteins
 Finally the precursor splits into three RNA components
 18S for small subunit of ribosome
 28S and 5.8 S for large subunit of ribosome
 The processing takes place in the nucleolus

Processing of mRNA
 The most extensively modified transcription product is the product of
RNA polymerase II
 The majority of this RNA will be processed to mRNA
 The primary transcript is some time called as pre-mRNA
 Cap is added at 5’
 Poly A tail added at 3’ end
 Splicing of pre-mRNA
 Editing of RNA
Addition of 5’ Cap

 The 5’ end of pre-mRNA contains A or G

 After synthesis the 5’ end of nascent RNA is immediately modified, First
a, phosphate is released by hydrolysis
 5’-5’ triphosphate linkage is formed between a GTP and 5’ end of RNA,
called Cap

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 Methylation at Cap 0, Cap 1 and Cap 2
Addition of Poly A tail

 Poly(A) is added at 3’ end by poly(A) polymerase after the cleavage of 3’

end at a specific sequence (AAUAAA), recognized by an endonulease
 The modification may increase the half-life of the mRNA, helps in
effective transport and translation
RNA Editing
• RNA editing changes the protein encoded by mRNA
 The sequence content of some mRNAs is altered after transcription
 RNA editing is the term for a change in the base sequence of RNA after
transcription by processes other than RNA splicing.
EXAMPLES
 Apolipoprotein B (apo B) plays an important role in the transportation of
triglycerols and cholesterol
 Apo B exist in two forms
 Apo B-100, 4563 aa long
 Apo B-48, 2152 aa of N-terminal of full protein
 The larger form, synthesized by the liver, participates in the transport of
lipids synthesized in our body cell

 These two forms are synthesized by a newly reported mechanism of

changing the nucleotide sequence of mRNA after transcription
 A specific cytidine residue of mRNA is deaminated to uridine, which
changes the codon at residue 2153 from CAA (Gln) to UAA (stop)
 The deaminase that catalyzes this reaction is present in the small intestine
but not in the liver, and is expressed only at certain developmental stage

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 A glutamate binding receptor in the vertebrate central nervous system
affects cation channel
 RNA editing changes a single glutamate codon (CAG) to arginine (CGG)
in this receptor
 Substitution of Gln by Arg in the receptor prevents Ca2+, but not Na+ from
flowing through the channel
 RNA editing is likely much more common than was previously
thought
 De-amination, that induces complex DNA-repair mechanism has
been used for generating molecular diversity at the RNA and hence
protein level
 In trypanosomes a different kind of RNA editing markedly changes
several mitochondrial mRNAs.
 Nearly half the uridine residues in these mRNAs are inserted by RNA
editing
 A guide RNA molecule identifies the sequence to be modified and
 A poly-U tail on the guide donates uridine residues to the mRNA
undergoing editing
Splicing of Pre-Mrna
 Most genes in higher eukaryotes are composed of exons and introns
 Introns must be spliced and exons linked to form the final mRNA in a
process called Splicing
 Comparison of intron sequences shows some highly conserved sequence
from yeast to mammals, begin with GU and end with AG
 The conserved sequences make three important sites called
 5’ splice site
 3’ splice site
Branch site
 In some forms of Thalassemia, defective synthesis of hemoglobin results
from a mutation leading to creation of new 3’ splicing site
 Mutations affecting splice sites have been estimated to cause 15% of all
genetic diseases
 In some forms of Thalassemia, defective synthesis of hemoglobin results
from a mutation leading to creation of new 3’ splicing site
 Mutations affecting splice sites have been estimated to cause 15% of all
genetic diseases


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
Spliceosomes
 Spliceosomes are large (60S), dynamic assemblies composed of
 snRNPs
 other proteins called splicing factors
 the mRNA precursors being processed
 Splicing begins with the recognition and pairing of the 5’ splice site of
intron in pre-mRNA by U1 snRNP by highly conserved hexa-nucleotides
sequence
 U2 snRNP then binds the branch site by base-pairing of its highly
conserved sequence
 Pre-assembled U4-U5 –U6 complex joins U1 and U2 complex & mRNA
precursor to form a complete spliceosome, coupled with ATP hydrolysis
 In this assembly U5 interacts with exon sequences in the 5’ splice site and
subsequently with the 3’ exon
 U6 disengages from U4 and base-pairs with U2, displacing U1 from the
spliceosome by interacting with the 5’end of the intron
 The U2-U6 helix is indispensable for splicing, suggesting that U2 and U6
snRNPs probably form the catalytic centre of the spliceosome
 U4 serves as an inhibitor that masks U6 until the specific splice sites are
aligned
 These rearrangements results in the first transesterification reaction,
generating the lariat intermediate and a cleaved 5’ exon
 Further rearrangements of RNA in the spliceosome facilitate the second
transesterification.
 The 5’ exon is aligned with 3’ exon such that the 3’ hydroxyl group of the
5’ exon is positioned to nucleophilically attack the 3’ splice site to
generate the spliced product.

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 U2, U5 and U6 bound to the excised lariat intron are released to complete
the splicing reaction
 Many steps in splicing process require ATP hydrolysis.
 RNA molecules play key roles in directing the alignment of splice sites
and in carrying out catalysis

Alternate Splicing
 Some pre-mRNA molecules can be spliced in alternative ways to yield
different mRNAs
 Alternative splicing is a widespread mechanism for generating protein
diversity
 The differential inclusion of exons into a mature RNA, alternative
splicing may be regulated to produce distinct forms of a protein for
specific tissues or developmental stages
 Alternative splicing provides a powerful mechanism for expanding the
versatility of genomic sequences

Self-Splicing
 The discovery of catalytic RNA was revealing in regard to both
mechanism and evolution
 RNAs form a surprisingly versatile class of molecules
 In Tetrahymena (a ciliated protozoan), a 414-nt intron is removed form a
6.4-kb precursor to yield the mature 26S rRNA
 Thomas Cech and his coworkers established that the RNA spliced itself to
precisely excise the 414-nt intron in the absence of any other protein, and
indeed have highly specific catalytic activity
 The self-splicing reaction requires an added guanosine nucleotide as
cofactor
 G binds to the RNA and then attacks the 5’ splice site to form a
phosphodiester bond with the 5’ end of the intron.
 This transesterification reaction generates a 3’-OH group at the end of the
upstream exon. This newly attached 3’-OH group then attacks the 3’
splice site.
 This second transesterification reaction joins the tow exons and leads to
the release of the 414-nt intron
 mRNA precursors in the mitochondria of yeast and fungi also undergo
self-splicing, as do some RNA precursors in the chloroplasts of
unicellular organisms such as Chlamydomonas

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  Self-splicing reaction can be classified according to the nature of the unit
that attacks the upstream splice site.
 Group I self-splicing is mediated by a guanosine cofactor, as in
Tetrahymena.
 Group II attacking moiety is the 2’-OH group of a specific adenylate of
the intron
 Group I and Group II self-splicing resembles spliceosome-catalyzed
splicing in two respect
 First, in initial step, a ribose hydroxyl group attacks the 5’ splice site, the
newly formed 3’-OH group of the upstream exon then attacks the 3’
splice site to form a phosphodiester bond with the downstream exon
 Second, both reactions are transesterification in which the phosphate
moieties at each splice site are retained in the products, the number of
phosphodiester bonds stays constant

 Lec# 4 TRANSLATION
Central Dogma of life
Genetic information is replicated from DNA to DNA
Genetic information is formulated into a message by the process of
Transcription
The transcribed message is decoded in the form of proteins through the process
of Translation
This flow is called Central Dogma of Life i.e.
Replication, Transcription and Translation

Proteins & Protein Synthesis

Proteins are very important being the workhorse of a cell
Protein Synthesis is more complex than either replication or transcription
Depends on both nucleic acid and protein factors.
Takes place on ribosomes enormous complexes containing rRNA and proteins
Transfer RNA (tRNAs), messenger RNA, and many proteins participate in
protein synthesis along with ribosomes.

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Implementation of Genetic Code
The Aminoacyl-tRNA synthetases implement the genetic code
Proteins are synthesized in the amino-to-carboxyl direction
Translation is a complex process, involving many steps and dozens of molecules
Basics of Protein Synthesis
The basics of protein synthesis are the same across all kingdoms of life
This indicates that the protein-synthesis system arose very early in evolution
Primarily emphasis will be on protein synthesis in prokaryotes with some
distinctive features in eukaryotes
RNAs & Protein Synthesis
RNAs involved in Protein Synthesis
tRNA rRNA mRNA

Transfer RNA
❖ Transfer RNA molecules have a Common Design
❖ The fidelity of protein synthesis requires the accurate recognition of
three-base codons on messenger RNA
❖ Each amino acid is represented by a three-letter codon on mRNA, but
cannot itself recognize a codon.
❖ Therefore, an amino acid is attached to a specific tRNA molecule that can
recognize the codon by Watson-Crick base pairing with mRNA
❖ Transfer RNA thus serves as the adapter molecule that binds to a specific
codon and brings with it a specific amino acid for incorporation into the
polypeptide chain
❖ tRNAs
❖ The number of tRNA molecules needs to be at least 20
❖ tRNA must be able to interact with
❖ aminoacyl tRNA synthetase
❖ mRNA
❖ Ribosomal sites like entry point, peptidyle transferase site etc.
❖ A protein factor to transport tRNA to ribosome

COMMON FEATURES OF tRNAs

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Robert Holley first determined the base sequence of yeast alanyl-tRNA
molecule in 1965, spending 7 long years
Sequences of hundreds of other tRNA molecules were determined soon,
revealing certain common feature like
1. Cloverleaf-like secondary structure in which about half the residues are
base-paires
2. Single chain containing 73-93 ribonucleotides (~25 kd)
3. Contain many unusual bases typically between 7 and 15

Some are methylated or dimethylated derivatives of A, U, C, and G

formed by enzymatic modification of a precursor tRNA, which
prevents the formation of certain base pairs
imparts hydrophobic character to some regions of tRNAs
4 About half the nucleotides in tRNAs are base-paired to form double
helices.
5 Five groups of bases are not base paired in this wayThe 3’ CCA
terminal region, a part of the acceptor stem
❖ The T C loop (ribothymine-pseudouracil-cytosine)
❖ The "extra arm" containing a variable number of residues
❖ The DHU loop, which contains several dihydrouracil residues
❖ The anticodon loop for interaction with mRNA

6 The 5’ end of a tRNA is phosphorylated and the 5’ terminal residue is

usually pG
7 The activated amino acid attaches to a 2’ or 3’ hydroxyl group of A
residue at the 3’ CCA end of the acceptor stem
8 The anticodon is present in a loop near the center of the sequence

3-D Structure of tRNA

First determined by Alexander Rich and Aaron Klug in 1974
Structure was determined by X-ray crystallography
The subject tRNA was yeast phenylalanyl-tRNA
Certain important properties of the tRNA structure were revealed by these
studies

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1 The molecule is L-shaped
2 There are two apparently continuous segments of double helix like A-
form DNA.
a) The base-pairing predicted from the sequence analysis is correct
b) Helix containing the 5’ and 3’ ends stacks on the helix that ends in the
T C loop to form one arm of the L-shaped structure
c) Remaining two helices stack to form the other arm of the structure

Most of the bases in the non-helical regions participate in hydrogen bonding

interactions
3 These interaction are not like those in Watson-Crick base pairs
4 The CCA terminus containing the amino acid attachment site extends
from one end of the L-shape
5 This single-stranded region can change conformation during amino acid
activation and peptide bond formation
6 The anticodon loop is at the other end of the L shape, making accessible
the three bases that make up the anticodon.
7 Thus architecture of the tRNA is well suited to its role

Linkage of amino acids to tRNA

➢ The amino acids arrive at the growing chain in activated form
➢ The linking of an amino acid to its corresponding tRNA is catalyzed by
an aminoacyl-tRNA synthetase e.g.
o glycyl-tRNA synthetase
o alanyl-tRNA synthetase
o cystenyl-tRNA synthetase and so on……
➢ ATP cleavage drives this activation reaction
➢ For each amino acid, there is usually one activating enzyme and at least
one kind of tRNA.
➢ The linkage is crucial for two reasons
o Activation of the amino acids
o Establishing the genetic code

Activation of Amino Acids

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 ➢ The activation reaction is catalyzed by specific aminoacyl-tRNA
synthetases, also called activating enzymes
➢ 1st step is the formation of an aminoacyl adenylate from an amino
acid and ATP (OH- of amino acid attack α-phosphate of ATP)
➢ -COOH of the amino acid thus gets linked to the phosphoryl group
of AMP; hence, it is also known as aminoacyl-AMP.
➢ The next step is the transfer of the aminoacyl group of aminoacyl-
AMP to a particular tRNA molecule to form aminoacyl-tRNA.
➢ These aminoacyl-tRNA are the amino acid ester, which are
activated intermediates in protein synthesis also called a charged
tRNA
➢ In amino acid esters, an amino acid is linked to either the 2’ or the
3’ hydroxyl group of the ribose unit at the 3’ end of tRNA

Accuracy in Translation
o The potential for error exists at each step
o The speed of translation is 20 amino acids/second
o Accuracy?
o The probability of errors (p) in a protein depends on
o n (the number of amino acid residues in a protein chain) and
o (the frequency of insertion of a wrong amino acid)
o Average protein chain size is about 300 aminoacids
o An error frequency of 10-2 would be intolerable
o Error value of 10-3 would lead to the error-free synthesis of a 300-
residue protein but not of a 1000-residue protein
o Thus, the error frequency must not exceed approximately 10-4
o Lower error frequencies will
▪ not dramatically increase the percentage of accurate proteins
▪ But, will reduce the rate of protein synthesis
o In fact, the observed values of are in the range of 10-4 to 10-5.
o Thus aminoacy-tRNA synthetase will incorporate the incorrect
amino acid only once in 104 or 105 catalytic reactions.

Recognition of Correct tRNA

Many synthetases recognize their tRNA partners primarily on the basis of their
anticodons

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Some other tRNA synthetases also use
acceptor stem and/or variable arm

Classification of Synthetases
Synthetases fall into two classes, class I and class II, each includes enzymes
specific for 10 of the 20 amino acids
❖ Glutaminyl-tRNA synthetase is a representative of class I
❖ Threonyl-tRNA synthetase is a representative of class II.

These two classes differ in many ways

o They bind to different faces of the tRNA molecule
o Class I enzymes acylate the 2’ -hydroxyl group of the terminal
adenosine of tRNA, whereas class II enzymes generally acylate
the 3’-hydroxyl group.
o The two classes bind ATP in different conformations.
o Most class I enzymes are monomeric, whereas most class II
enzymes are dimeric
Translation part 2
Lec# 5 TRANSLATION 2
Ribosome
1 Ribosome coordinate the interplay of charged tRNAs & mRNA, leading to
protein synthesis
2 The ribosome is a ribozyme; strongly supporting the notion that the ribosome
is a surviving inhabitant of the RNA world
3 E. coli ribosome is a ribonucleoprotein assembly with a mass of about 2700
kd
a diameter of approximately 200 Å
sedimentation coefficient 70S
4 The 20,000 ribosomes in a bacterial cell constitute nearly 25% of its dry mass
Two subunits: a large subunit (50S) and a small subunit (30S
The 50S subunit contains
❖ 34 different proteins (L1 through L34)
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 ❖ two rRNA molecules, a 23S and a 5S species

The 30S subunit contains

❖ 21 different proteins (referred to as S1 through S21)
❖ 16S rRNA molecule

5 A ribosome contains
one copy of each of the rRNA molecule (23S, 5S, & 16S)
two copies each of the L7 and L12 proteins
one copy of each of the other proteins
Only one protein is common to both subunits: S20/L26
6 Both the 30S and the 50S subunits can be reconstituted in vitro (Nomura
1968) from their constituent proteins and RNA
7 This reconstitution is an outstanding example of the principle that supra-
molecular complexes can form spontaneously from their macromolecular
constituents

Ribosomal RNAs
❖ Ribosomal RNAs (23S, 5S, and 16S rRNA) play a central role in protein
synthesis
❖ RNAs constitutes nearly two-third of the mass of ribosomes
❖ The presence of all of the three RNAs (23S, 5S, and 16S rRNA) is critical
for ribosomal architecture and function
❖ rRNAs are formed by cleavage of primary 30S transcripts and further
processing
❖ By comparing the nucleotide sequences of many species, in combination
with chemical modification and digestion experiments
❖ The base-pairing patterns was deduced
❖ Conserved features have been detected
❖ The striking finding is that ribosomal RNAs (rRNAs) are folded into
defined structures with many short duplex regions
▪ For years, ribosomal proteins were presumed to control protein synthesis
and ribosomal RNAs were presumed to play structural role
▪ The current view is almost the reverse
▪ The detailed structures make it clear that the key sites in the ribosome are
composed almost entirely of RNA
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 ▪ Recent investigations show that 2’-OH of A2451 plays catalytic role
(peptidyl transferase) in peptide bond formation
▪ So the almost inescapable conclusion is that ribosome initially consisted
only of RNA (catalytic component) and that the proteins (co-factor
component) were added later during evolution

mRna
• The start signal is AUG (or GUG) preceded by several bases that pair
with 16s rRNA
• Comparison of mRNA and Protein sequence show that translation does
not begin immediately at the 5’ terminus of mRNA
• First translated codon is ≥25 nucleotides away from the 5’ end
• In fact, all known mRNA molecules contain signals that define the
beginning (initiation codon) and end (termination codon) of each
encoded polypeptide chain
• Even in polycistronic, or polygenic mRNAs each protein has its own start
and stop signals
• Also established by the fact that nearly half the amino-terminal residues
of mature proteins in E. coli carry methionine
o Are there any additional signals necessary to specify a translation
start site?
o The answer is, Yes!
o Nuclease protection assay/foot printing of the initiation complexes
revealed that about 30 nucleotides of mRNA at 5’ end were
protected from digestion.
❖ As expected, each initiator region displays an AUG
❖ Each initiator region has a purines-rich sequence centered ~10nt upstream
the initiator codon called “Shine-Dalgarno sequence”
❖ The 3’ end of 16S rRNA present in 30S subunit contains a sequence that
is complementary to the Shine-Dalgarno sequence in mRNA
❖ Mutagenesis of the CCUCC (GGAGG in Shine-Dalgarno) sequence near
the 3’ end of 16S rRNA to ACACA markedly interferes with the
recognition of start sites in mRNA

o These observations show that the initiator region of mRNA binds to the
16S rRNA very near to its 3’ end

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 o The number of base pairs linking mRNA and 16S rRNA ranges from
three to nine
o Thus, two kinds of interactions determine where protein synthesis
startsthe pairing of mRNA bases with the 3’ end of 16S rRNA and

pairing of the initiator codon on mRNA (AUG) with the anticodon of an

initiator tRNA molecule

Stages in Protein Synthesis

▪ There are three stages in protein synthesis

Initiation Elongation Termination

Maturation
o Post-translational modifications
o Chemical modifications
o Truncation

Initiation of Protein Synthesis

➢ Protein synthesis begins with the interaction of the 30S subunit and
mRNA through the Shine-Delgarno sequence
➢ On formation of this complex, the initiator tRNA charged with formyl-
methionine binds to the initiator AUG codon
➢ This is followed by binding of 50S subunit to the 30S subunit to form the
complete 70S ribosome
➢ Generally protein synthesis in bacteria is initiated by using formyl-
methionine as first amino acid at N-terminal
➢ A special tRNA called initiator tRNA brings f-Met to the ribosome
(tRNAf)
➢ Actually tRNAm is converted by a specific enzyme into tRNAfThe
activated formyl donor in this reaction is N10-formyltetrahydrofolate
➢ The tRNAf differs from the usual tRNA (tRNAm) that inserts methionine
in internal positions
➢ In approximately half of E. coli proteins,
N-formylmethionine is removed when the nascent chain is about 10
amino acids long

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 Elongation
➢ The three sites on 70S ribosome provide clues
➢ The initiator tRNA is bound in the P site on the ribosome
➢ A charged tRNA with an anticodon complementary to the codon in the A
site then binds
➢ The peptidyl transferase center includes residues that promote this
reaction by
➢ orienting -NH2 group on the A site aminoacyl-tRNA such that it can
attack carbonyl group of the P site aminoacyl-tRNA
➢ The catalytic role seems to be played by the 2’-OH of A2451 of the 23S
rRNA
➢ helping to stabilize the tetrahedral intermediate that forms
➢ Sequential addition of amino acids takes place on the carboxyl end of the
growing peptide chain

tRNA-Binding Sites on Ribosomes

• Structure of the 70S ribosome bound to a fragment of mRNA
revealed three tRNAs bound to it
• mRNA fragment is bound within the 30S
• Each of the tRNA molecules bridges between the 30S and 50S
subunits
• At the 30S end, two of the three tRNA molecules are bound to the
mRNA fragment through anticodon-codon base pairs
• .These binding sites are called the A sites (for aminoacyl) and the P
site (for peptidyl) The third tRNA molecule is bound to an adjacent
site called the E site (for exit)
• The other end of each tRNA molecule interacts with the 50S subunit
• The acceptor stems of the tRNA molecules occupying the A site and
the P site converge at a site where a peptide bond is formed
• A tunnel connects this site to the back of the ribosome through which
the polypeptide chain passes through this tunnel during synthesis

Translocation
For translation to proceed, the mRNA must be moved (or translocated) so that
the codon for the next amino acid to be added is located in the A site

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 ➢ This translocation takes place through the action of a protein
enzyme called elongation factor G (EF-G)
➢ Why the amino terminus of the initial methionine molecule is
modified by the attachment of a formyl group?
➢ The answer is; To prevent attack of amino group of first amino
acid on its own carbonyl group

Codon-Anticodon Interaction
➢ Does the amino acids attached to the tRNA play any role in this process?
➢ The attached cysteine unit was converted into alanine by adding
Raney nickel to Cys-tRNACys
➢ The result was a mischarged aminoacyl-tRNA (Ala-tRNAcys)
➢ In recent years, the ability of mischarged tRNAs to transfer their
amino acid cargo to a growing polypeptide chain has been used to
synthesize peptides with amino acids not found in proteins
➢ More than 100 different unnatural amino acids have been incorporated
in this way
➢ However, only l-amino acids can be used; apparently this
stereochemistry is a prerequisit for peptide-bond formation to take
place

Wobble Hypothesis
➢ According to Watson-Crick pairing, a particular anticodon can
recognize only one codon
➢ The facts are otherwise. It is determined experimentally that
some pure tRNA molecules can recognize more than one codon
e.g. the yeast alanyl-tRNA binds to three codons: GCU, GCC,
and GCA.
➢ Pairing between first two bases of a codon with anticodon
follows Watson-Crick principal, whereas
➢ According to the wobble hypothesis, allowed pairings at the
third base of the codon is not necessarily Watson-Crick pairing
➢ Assumption of this steric freedom in the pairing of the third
base of the codon is called "Wobble Hypothesis”
➢ The wobble hypothesis is now firmly established
➢

30
 Allowed pairings at the third base of the codon according to the wobble
hypothesis
First base of anticodon Third base of codon
C G
A U
U A or G
G U or C
I U, C, or A

❖ Two generalizations concerning the codon-anticodon interaction can be

made:
❖ The first two bases of a codon pair in the standard way. Hence, codons
that differ in either of their first two bases must be recognized by different
tRNAs
❖ The first base of an anticodon determines whether a particular tRNA
molecule reads one, two, or three kinds of codons: C or A (one codon), U
or G (two codons), or I (three codons).
❖ The inosine in tRNA is formed by post-transcriptional oxidative de-
amination of adenosine

Why is wobble tolerated in the third position of the codon but not in the first
two?
❖ The 16S rRNA subunit has two adenine bases that form hydrogen bonds
on the minor-groove side of the codon-anticodon duplex and does not
allow incorrect pairing
❖ No such inspection device is present for the third position
❖ The fact again indicates that ribosome plays an active role in decoding the
codon-anticodon interactions

Termination
❖ An open reading frame starts from the end of 5’ UTR and continues
until a stop codon
• Stop codon are UAA UAG UGA
❖ Stop codon do not have any corresponding tRNA
❖ This results in release of protein

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 Lec# 6 Post translational Modification

Post-translational modifications refer to any alteration in the amino

acid sequence of the protein after its synthesis.
• It may involve modifying the amino acid side chain, terminal amino or
carboxyl group using covalent or enzymatic means
following protein biosynthesis.
• Generally, these modifications influence the structure, stability,
activity, cellular localization, or substrate specificity of the protein.
• The post-translational modification provides complexity to the
proteome for diverse functions with a limited number of genes.

Location
Post-translational modifications (PTMs) mainly occur in the endoplasmic
reticulum of the cell but sometimes continue in the Golgi bodies as well.

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After synthesis is completed, proteins can be modified by various methods
such as phosphorylation, glycosylation, ADP ribosylation, hydroxylation,
and addition of other groups.
Types of post-translational modification

There are many typs of protein modification, which are mostly catalyzed by
enzymes that recognize specific target sequences in proteins. These modifications
regulate protein folding by targeting specific subcellular compartments, interacting
with ligands or other proteins, or by bringing about a change in their functional state
including catalytic activity or signaling. The most common PTMs are:

Based on Chemical groups

Phosphorylation

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Reversible phosphorylation of proteins involves addition of a phosphate group on
serine, threonine, or tyrosine residues and is one of the important and extensively
studied PTM in both prokaryotes and eukaryotes.

Several enzymes or signaling proteins are switched ‘on’ or ‘off’ by phosphorylation or

dephosphorylation. Phosphorylation is performed by enzymes called ‘kinases’, while
dephosphorylation is performed by ‘phosphatases’.

Addition of a phosphate group can convert a previously uncharged pocket of protein

into a negatively charged and hydrophilic protein thereby inducing conformational
changes in the protein.

Phosphorylation has implications in several cellular processes, including cell cycle,

growth, apoptosis and signal transduction pathways. One example is the activation
of p53, a tumor suppressor protein. p53 is used in cancer therapeutics and is
activated by phosphorylation of its N-terminal by several kinases.

Acetylation

Acetylation refers to addition of acetyl group in a protein. It is involved in several

biological functions, including protein stability, location, synthesis; apoptosis; cancer;
DNA stability. Acetylation and deacetylation of histone form a critical part of gene
regulation.

Acetylation of histones reduces the positive charge on histone, reducing its

interaction with the negatively charged phosphate groups of DNA, making it less
tightly wound to DNA and accessible to gene transcription. Acetylation of p53, a
tumor suppressor gene, is crucial for its growth suppressing properties.

Hydroxylation

This process adds a hydroxyl group (-OH) to the proteins. It is catalyzed by enzymes
termed as ‘hydroxylases’ and aids in converting hydrophobic or lipophilic compounds
into hydrophilic compounds.

Methylation

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Methylation refers to addition of a methyl group to lysine or arginine residue of a
protein. Arginine can be methylated once or twice, while lysine can be methylated
once, twice, or thrice. Methylation is achieved by enzymes called
methyltransferases. Methylation has been widely studied in histones wherein histone
methylation can lead to gene activation or repression based on the residue that is
methylated.

Based on Complex groups

Glycosylation

Glycosylation involves addition of an oligosaccharide termed ‘glycan’ to either a

nitrogen atom (N-linked glycosylation) or an oxygen atom (O-linked glycosylation). N-
linked glycosylation occurs in the amide nitrogen of asparagine, while the O-linked
glycosylation occurs on the oxygen atom of serine or threonine.

Carbohydrates present in the form of N-linked or O-linked oligosaccharides are

present on the surface of cells and secrete proteins. They have critical roles in
protein sorting, immune recognition, receptor binding, inflammation, and
pathogenicity. For example, N-linked glycans on an immune cell can dictate how it
migrates to specific sites. Similarly, it can also determine how a cell recognizes ‘self’
and ‘non-self’.

AMPylation

AMPylation refers to reversible addition of AMP to a protein. It involves formation of

a phosphodiester bond between the hydroxyl group of the protein and the phosphate
group of AMP.

Lipidation

The covalent binding of a lipid group to a protein is called lipidation. Lipidation can be
further subdivided into prenylation, N-myristoylation, palmitoylation, and
glycosylphosphatidylinositol (GPI)-anchor addition.

35
Prenylation involves the addition of isoprenoid moiety to a cysteine residue of a
substrate protein. It is critical in controlling the localization and activity of several
proteins that have crucial functions in biological regulation.

Myristoylation involves the addition of myristoyl group to a glycine residue by an

amide bond. It has functions in membrane association and apoptosis. In
palmitoylation, a palmitoyl group is added to a cysteine residue of a protein.

In GPI-anchor addition, the carboxyl-terminal signal peptide of the protein is split and
replaced by a GPI anchor. Recent research in human genetics has revealed that GPI
anchors are important for human health. Any defects in the assembling, attachment
or remodeling of GPI anchors lead to genetic diseases known as inherited GPI
deficiency.

Based upon addition of Polypeptides

Ubiquitination

Ubiquitination involves addition of a protein found ubiquitously, termed ‘ubiquitin’, to

the lysine residue of a substrate. Either a single ubiquitin molecule
(monoubiquitination) or a chain of several ubiquitin molecules may be attached
(polyubiquitination).

Polyubiquitinated proteins are recognized by the 26S proteasome and are

subsequently targeted for proteolysis or degradation. Monoubiquitinated proteins
may influence cell tracking and endocytosis.

Based upon Protein cleavage

Proteolysis

Proteolysis refers to breakdown of proteins into smaller polypeptides or amino acids.

For example, removal of N-terminal methionine, a signal peptide, after translation
leads to conversion of an inactive or non-functional protein to an active one.

Based upon Amino acid modification

Deamidation
36
Deamidation is the removal or conversion of asparagine or glutamine residue to
another functional group. Asparagine is converted to aspartic acid or isoaspartic
acid, while glutamine is converted to glutamic acid or pyroglutamic acid. This
modification can change the protein structure, stability, and function.

Significance
Proteins are synthesized by ribosomes translating mRNA into polypeptide
chains, which may then undergo modifications to form the mature protein
product.
Post-translational modifications of proteins, which are not gene- template
based, can regulate the protein functions, by causing changes in protein
activity, their cellular locations and dynamic interactions with other
proteins.
PTMs have significant biological functions which include:
• Aids in proper protein folding – few lectin molecules called calnexin
binds to glycosylated proteins and assist in its folding.
• Confers stability to the protein- glycosylation can modify the stability
of the protein by increasing protein half life.
• It protects the protein against cleavage by proteolytic enzyme by
blocking the cleavage sites.
• Protein sorting or translocation- If phosphorylated mannose residues
are present in the protein it always goes to lysosome.
• It regulates protein activity and function- phosphorylation of protein
is a reversible PTM which activates the protein.
• Acetylation regulates many diverse functions, including DNA
recognition, protein-protein interaction and protein stability.
• Redox-dependent PTM of proteins is emerging as a key signaling
system conserved through evolution, influences many aspects of
cellular homeostasis.
• PTMs are important components in cell signaling, as for example
when prohormones are converted to hormones.
• It significantly increases the diversity and complexity in the proteome.

37