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Lecture 10-12 Protein Synthesis PDF

The document discusses protein synthesis and gene regulation in prokaryotes. It describes the genetic code, how it was deciphered, the central dogma of molecular biology, transcription and translation. It also discusses different mechanisms of gene regulation including operons, repressors, inducers and attenuation.

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Vaibhav Kumar
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40 views

Lecture 10-12 Protein Synthesis PDF

The document discusses protein synthesis and gene regulation in prokaryotes. It describes the genetic code, how it was deciphered, the central dogma of molecular biology, transcription and translation. It also discusses different mechanisms of gene regulation including operons, repressors, inducers and attenuation.

Uploaded by

Vaibhav Kumar
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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Lectures 10-12

Protein Synthesis
Deciphering the Genetic Code
v 1865 –Mendel defined the basic unit of inheritance as the gene
v 1900 –Mendel’s forgotten work resurfaces; nature of gene is still
unknown
v 1944 –it is established that a gene is made of DNA
v 1953 –Watson-Crick’s double helix structure for DNA
DNA: L = {A, C, G, T}
One big question
RNA: L = {A, C, G, U}
remained unanswered:
Double Stranded DNA how is the information
5’ A T T G C C C A T 3’
in the DNA strand
3` T A A C G G G T A 5’ translated to protein?

George Gamow and the “RNA tie Club”
v Brotherhood consisted of 20 regular members,
one for each amino acid
v Watson was PRO (proline)
v Four honorary members, one for each
nucleotide
v Eight of these members were or became Nobel
Laureates Georgiy Antonovich Gamov
March 4, 1904- August 19, 1968
Big Bang Theory
Formation of stars
Some of the Ideas Proposed
by the RNA Club

v The Adapter Hypothesis by Francis Crick – some


unknown biological entity carried the amino acids and
put them in the sequence order
v Gamow proposed that a three-letter code would be
sufficient to define all 20 amino acids
Combinatorial Figures of Gamow’s
proposition

v Number of diamonds where top and


bottom are identical
v 4C1×2 =8
v Number of diamonds where top and
bottom are different
v 4C2×2 =12
Analysis Presented by Crick
v Genetic code is in triplets (codon)
v There are 20 amino acids
v There are 4 alphabets A, T, G, C
v 4 & 42< 20, ∴43 = 64 (but with redundancies ?)
v The genetic code should be comma free
v Only one valid reading frame
v [abc][def][ghi][jkl]
v NOT a [bcd][efg]hij]…
v NOT ab[cde][fgh][ijk]…
v AAA, TTT, GGG, CCC are not possible because for example AAAAAA the reading frame
is ambiguous
v That leave 64-4 = 60
v ATGATG must be read unambiguously, So, whenever ATG is a codon, TGA or GAT is
not
v That gives (1/3)*60 = 20
Marshall Nirenberg Deciphered
the Genetic Code in 1961

Marshall Warren Nirenberg


April 10, 1927 – January 15,
2010, ; Jewish American
biochemist and geneticist

M. W. Nirenberg and P. Leder, 1964, Science 145:1399


The Genetic Code

The genetic
code is a map of
Codons “C” to
Amino Acids “A”
g: C à A
Grouping by Physical Properties of Amino
Acids Best Explains the Genetic Code Table
Important points related to translation
v The particular amino acid sequence of a protein is constructed through the
translation of information encoded in mRNA. This process is carried out by
ribosomes.
v Amino acids are specified by mRNA codons consisting of nucleotide
triplets. Translation requires adaptor molecules, the tRNAs, that recognize
codons and insert amino acids into their appropriate sequential positions
in the polypeptide.
v The base sequences of the codons were deduced from experiments using
synthetic mRNAs of known composition and sequence.
v  The codon AUG signals initiation of translation. The triplets UAA, UAG,
and UGA are signals for termination.
Degeneracy of the Genetic Code
The Wobble Hypothesis

v Alignment of the two RNAs is


antiparallel. The tRNA is shown in
the traditional cloverleaf
configuration
v Three different codon pairing
relationships are possible when the
tRNA anticodon contains inosinate.
Five Stage of Protein Synthesis in E. coli
The Ribosome
Aminoacyl t-RNA Synthetase

Aminoacyl-tRNA Synthetases
Attach the Correct Amino Acids to
Their tRNAs
v aminoacyl-tRNA synthetases
esterify the 20 amino acids to their
corresponding tRNAs. Each
enzyme is specific for one amino
acid and one or more
corresponding tRNAs
v  Proofreading by Aminoacyl-tRNA
Synthetases
v  Interaction between an
Aminoacyl-tRNA Synthetase and a
tRNA
Stage 2: A Specific Amino Acid Initiates
Protein Synthesis
v Although methionine has only one codon, (5’)AUG, all organisms have two
tRNAs for methionine
v One is used exclusively when (5’)AUG is the initiation codon for protein
synthesis
v The other is used to code for a Met residue in an internal position in a
polypeptide
v The amino acid incorporated in
v response to the (5’)AUG initiation codon is N-formylmethionine (fMet)

Methionine + tRNAfMet + ATP à Met-tRNAfMet + AMP + PPi


Stages of protein
synthesis
Regulation of Gene
Expression
Genes are expressed when required
v Some proteins are expressed abundantly such as elongation factors
and rubisco
v Others such as DNA repair enzymes are synthesized very few in
number
v Requirements of gene products varies in the cell-type and in its life
cycle
v Ribosomes are synthesized rapidly during the exponential growth phase of
the cell
What factors determine the cellular
concentration of proteins
1.  Synthesis of the primary RNA transcript (transcription)
2.  Posttranscriptional modification of mRNA
3.  Messenger RNA degradation
4.  Protein synthesis (translation)
5.  Posttranslational modification of proteins
6.  Protein targeting and transport
7.  Protein degradation
Gene regulation

House Keeping
Regulated Genes
Genes
• Constitutive gene • Inducible gene
expression expression
• Repressible gene
expression
Representative Prokaryotic Operon

v Genes A, B, and C are transcribed on one polycistronic mRNA. Typical


regulatory sequences include binding sites for proteins that either
activate or repress transcription from the promoter
RNA polymerase

v RNA polymerases bind to DNA and initiate transcription at promoters, sites


generally found near points at which RNA synthesis begins on the DNA template
v The regulation of transcription initiation often entails changes in how RNA
polymerase interacts with a promoter
Negative Regulation of Gene Expression
Positive Regulation of Gene Expression
Lactosde metabolism in E. coli
The Lac Operon

v The I gene encodes the Lac repressor. v O1 is the main operator for the lac
The lac Z, Y, and A genes encode beta- operon
galactosidase, galactoside permease, v The Lac repressor binds to the main
and thiogalactoside transacetylase, operator and O2 or O3, apparently
respectively forming a loop in the DNA that might
wrap around the repressor
The Lac Operon
The Trp Operon This operon is regulated by two
mechanisms:
v When tryptophan levels are
high, the repressor binds to its
operator
v Transcription of trp mRNA is
attenuated
The Trp mRNA Sequence
What happens at high Tryptophan levels

v When tryptophan levels are high, the ribosome quickly translates sequence 1 (open reading frame encoding
leader peptide) and blocks sequence 2 before sequence 3 is transcribed. Continued transcription leads to
attenuation at the terminator-like attenuator structure formed by sequences 3 and 4
What happens at low Tryptophan levels

v When tryptophan levels are low, the ribosome pauses at the Trp codons in sequence 1. Formation of the
paired structure between sequences 2 and 3 prevents attenuation, because sequence 3 is no longer
available to form the attenuator structure with sequence 4. The 2:3 structure, unlike the 3:4 attenuator,
does not prevent transcription.

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