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Transcript
Protein Synthesis
(Sections 5.1 to 5.4)
 Inherited instructions stored in DNA direct the production of
proteins
A Brief History
 Gregor Mendel = factors are responsible for inherited traits
How genes control metabolism?
1. One gene – one enzyme
 Archibald Garrod (1909) = hypothesis states “one gene
 Cells synthesize and degrade organic compounds via
metabolic pathways, with each sequential step catalyzed
by a specific enzyme (figure 2, p. 234)
 Beadle and Tatem experiment (3o years later) were able
to demonstrate this experimentally
i. Neurospora (bread mold) - can survive with minimal
nutrients, however some mutants were not able to
survive why?
ii. Looked at metabolic pathway for the synthesis of
arginine- they distinguished mutants by which
substance needed to be added to the medium in
order for the mold to grow
Precursor
enzyme A
ornithine
enzyme B
citrulline
enzyme C
arginine enzyme D arginine
Succinate
iii. Conclusion: each mutant lacked a different
functional enzyme, thus blocked at different parts
of the metabolic pathway (see fig. 2, p. 235)
2. One gene – one polypeptide
 Today, we understand that genes code for proteins, and not
all proteins are enzymes. Also, some proteins consist of
more that one polypeptide, each peptide coded by a different
gene (Vernon Ingram) see p. 235
The Central Dogma
The central dogma of molecular biology (Francis Crick,1958)
 The central dogma of molecular biology deals with the
detailed residue-by-residue transfer of sequential
information.
 It states that information cannot be transferred back from
protein to either protein or nucleic acid. DNA  RNA  protein
 DNA cannot leave the nucleus (keeps it from being damaged)
An Overview of Protein Synthesis
 RNA links the genetic code stored in DNA to the production
of Proteins – RNA copies the code in DNA (transcription)
and translates the message into a polypeptide chain
 DNA structure vs RNA structure:
o
o
o
o
DNA
DNA
DNA
DNA
double-stranded, RNA single-stranded
has thymine, RNA has uracil
has Deoxyribose sugar, RNA has ribose sugar
is the genetic code, RNA interprets the code
The Flow of genetic information:
 The linear sequence of nucleotides in DNA ultimately
determines the linear sequence of amino acid in a protein.
DNA
transcription
RNA
RNA processing
RNA
translation
polypeptide
Protein Synthesis occurs in Two Stages:
1. Transcription = the synthesis of messenger RNA (mRNA)
using DNA as a template
 Resulting mRNA strand carries this transcript
(small strand) to the protein-synthesizing
machinery
2. Translation = synthesis of a polypeptide, which occurs
under the direction of mRNA
 Linear sequence of bases in mRNA is translated
into the linear sequence of amino acids
 Translation occurs at protein-synthesizing
machinery, which consists of ribosomes,
ribosomal RNA (rRNA), and proteins that facilitate
the addition of amino acids to form polypeptide
NOTE: ****Prokaryotes and eukaryotes differ in how protein
synthesis is organized within their cells.
 In Prokaryotes – happens in rapid successions (no nucleus)
 In Eukaryotes – mRNA is modified prior to leaving the
nucleus
Ribonucleic Acid - RNA
 There are three major classes of RNA:
o Messenger RNA (mRNA) – hold code
o Transfer RNA (tRNA) – help in carrying amino acids
to ribosomal complex
o Ribosomal RNA (rRNA) – structural component of
ribosomal complext
Transcription – In General
 Flow of information from gene to protein is based on a
triplet code (p. 240)
o Recall, there are only 4 different types of nucleotide,
but there are 20 different amino acids. How can we
code for all these amino aicds?
o RNA is read 3 nucleotide bases at a time = a codon
Ex. AUG = methionine – start codon / UUA, UAG, UGA = stop
o The ability to extract the intended message depends
on how the code is read
o The reading frame dictate that this is done 3 bases at
a time with no overlap ex. THE RED DOG
 Genes are first transcribed into mRNA:
o For each gene, only one of the two DNA strands
(template strand) is transcribed
o The strand that is not transcribed is called the parent
strand
o Which strand serves as the template stand varies for
each gene
 mRNA stand is complimentary to DNA template strand
o Recall, uracil (U) in RNA is used in place of thymine
(T)  ie. U pairs with A
Translation – In General
 Linear sequence of codons in mRNA is translated into linear
sequence of amino acids in polypeptide
o Each mRNA codon specifies which amino acid is
incorporated next into growing polypeptide
o Thus, the number of nucleotides is 3 times > a.a.
Cracking the Genetic Code
The first codon was deciphered in 1961 by Nirenberg of the
NIH
 61 of 64 triplets code for amino acids
 AUG is the start signal for translation and codes for
methionine
 UAA, UAG, UGA code only for signal termination = stop
codon
 There can be two or more codons for each amino acid (ex. UUU,
UUC both code for phenylalanine)
 Codons only code for one amino acid (ex. UUU only codes for
phenylalanine)
 The correct ordering (called the reading frame) and
grouping of nucleotides is important in the molecular
language of cells. A change in the reading frame leads to a
change in the peptide sequence!  mutation
Proteins Synthesis – The Specifics!
A. Transcription = synthesis of mRNA



transcription of mRNA is catalyzed by RNA polymerase
(separates DNA and builds mRNA in 5’ to 3’ direction)
specific DNA nucleotide sequences mark where
transcription of a gene begins (initiation) and ends
(termination).
The nucleotide sequence that is transcribed into mRNA
by RNA polymerase is called a transcription unit.
1. Binding of RNA polymerase =


RNA poly. Binds to specific DNA region called the
promoter region  marks initiation
In eukaryotes, the promoter region is approx. 100
nucleotides long and consists of:
i. Promoter region
ii. Binding site – where DNA binding
proteins attach



DNA binding proteins (aka transcription factors) =
bind to TATA box just upstream to initiation site
TATA Box = short repeating nucleotide sequence (~25)
upstream to promoter region (initiation site)
RNA polymerase recognizes transcription factors and
binds to promoter region.
2. Elongation of RNA Strand =



RNA polymerase continues to unwind DNA and add
RNA nucleotides in 5’ to 3’ direction
mRNA strand grows about 30-60 nucleotides/second
several molecules of RNA polymerase can
simultaneously transcribe the same gene!
3. Termination of Transcription =


transcription continues until RNA polymerase
transcribes a terminator sequence
in eukaryotes the most common terminator sequence is
AAUAA
4. Modification of RNA Transcript – Eukaryotes only!



Primary transcript = general term for initial RNA
strand transcribed from DNA
Pre-mRNA = primary transcript that will be processed
to functional mRNA
Transcript can be processed in two ways:
a. alteration of both the 3’ and 5’ ends
- 5’ CAP = guanosine triphosphate is added
(protects the mRNA strand from degradation,
helps in initiation of translation
- 3’ END = poly A tail (protects mRNA from
degredation, facilitates attachment to ribosomal
complex, assists in export of nucleus)
b. removal of intervening sequences
- coding regions (exons) are interrupted by non
coding regions (introns)
- during processing introns are removed by
splicosomes, and exons are joined (RNA splicing)
are removed before mRNA leaves the nucleus
- small nuclear ribonucleoproteins (SnRNPs) play a
key role in RNA splicing
Why have introns?
 Intron DNA sequences may control gene activity
 The splicing process may help regulate the export of mRNA
 Introns may allow a single genet to direct the synthesis of
different proteins (i.e if the same RNA transcript is processed
differently)
B. Translation = synthesis of peptide, coordinated by mRNA
We Need:
mRNA
tRNA
amino acids
polypeptide
ribosomes
protein
Ribosomes:
 are made up of two subunits (60% rRNA and 40% protein)
that hold the mRNA in place in order for translation to
occur.
 Each ribosome has three biding sites:
 P site = holds tRNA carrying growing peptide
 A site = holds tRNA carrying next amino acid
 E site = where tRNA exits from
Transfer RNA, tRNA:
 RNA strand (~80 bp) transcribed from DNA in nucleus
 3D shape held together by H-bonds
 can be used repeatedly during translation
 proteins are synthesized according to the sequence of codons,
tRNA helps in translation of RNA code to a.a sequence. How?
 tRNA aligns the appropriate amino acid by:
- transfers amino acid from cytoplasm to ribosomal
complex
- each tRNA strand has an amino acid attachment
site at one end and a anticodon at the opposite
end.
- Anticodon is complimentary to the RNA codon for
that amino acid
- The attaching of the a.a. to its tRNA is catalyzed
by a specific aminoacyl-tRNA synthetase
(required ATP)  each amino acid has its own
synthetase
Building a polypeptide – Occurs in Thee Stages
1. Initiation of Translation
 Small (40) subunit of ribosome binds to mRNA strand (5’ cap
of mRNA helps binding)
 Initiator Met-tRNA (start anticodon) finds AUG (start codon)
downstream on mRNA and binds to it, large subunit then
binds to initiator tRNA forming initiation complex (small
ribosome unit, mRNA and met-tRNA)
 Large ribosomal subunit (60) attached to small one; initiator
tRNA fits into P-site of ribosome  vacant A site is ready for
next amino acid
2. Elongation
 Codon recognition: mRNA codon in A site of ribosome forms
H-Bonds with anticodon of entering tRNA carrying the next
amino acid
 Peptide bond formation: a peptide bond is formed between
growing polypeptide in P-site and new amino acid in A-site
by peptidyl transferase  polypeptide is transferred to Asite
 Translocation (ribosome moves forward one codon): the tRNA
that was in the P-site is released (E-site). The tRNA in the Asite is translocated to the P-site (taking growing peptide with
it)  this repeats along the length of mRNA …..
3. Termination
 Occurs when a stop codon is reached (UGA,UAG, or UAA)
 Stop codons do not code for any amino acid
 Reaching stop sequence causes a release factor or bind to
the A-site of the ribosome and facilitates the release of the
polypeptide.
 Small and large ribosomal units separate
 Polypeptide is now ready for further processing and folding!
Recall,
 A protein’s sequence (primary structure) determines how the
peptide will coil and fold into its 3D shape
 Some proteins undergo post-translational modifications
before they become fully functional (ie chemical
modifications, or chain length modifications)
Multiple Roles of RNA in the cell
 RNA has many other critical roles in the cell:
1. information carrier = mRNA carries genetic info form
DNA to ribosomes
2. adaptor molecule = tRNA translate info from mRNA
into protein SRP RNA directs the translation complex to
ER
3. catalysts and structural molecule – rRNA plays
structural and enzymatic role in ribosomes; snRNA
catalyzses RNA splicing
4. viral genomes – some viruses use RNA as genetic
material
Control Mechanisms
(Section 5,5)

Not all of our 42,000 genes are needed at all times, so
transcription factors turn genes on and off as required

Note: some housekeeping genes are always being transcribed
since cells need these at all times.

In eukaryotes, there are 4 levels of gene control (expression):
i)
transcriptional,
ii)
posttranscriptional,
iii)
translational, and
iv)
iv) posttranslational.
See table 1.p 255  know them!
Operons

Operons = are clusters of genes under the control of one
promoter and one operator  ex lac operon, trp operon
Lac operon
o
Form of control only used in bacteria
o
In bacteria such as E.coli found in the digestive
system of mamals, β-galactosidase is the enzyme
responsible for breaking down lactose (enzyme is not
always required)
o
β-galactosidase is part of an operon
o
lacl protein binds to operon blocking transcription
o
in the presence of lactose a repressor protein (lacl
protein) normally bound to operon leaves and binds
to lactose  transcription of the lac operon no longer
blocked (no enzymes are made)  see fig. 2 p. 256
o
Why? When there is no milk, no enzyme is needed
The trp Operon

Works in the opposite manner to the lac operon

tryptophan is an amino acid used by bacteria to produce
proteins, when available in its environment bacteria stop
producing tryptophan and absorb it from its environment

operon acitivity is inhibited when the concentration of
tryptophan in the environment increases  tryptophan
binds to operator region of operon  see fig. 3, p. 257