Lecture 15: Translation Flashcards
Once an mRNA has been produced by transcription process, the information
present in its nucleotide sequence is used to
synthesize a protein.
Translation is a process of converting nucleic acid codes into
amino acid sequences
Coding sequences (open reading frame) is the
translated region, while the untranslated regions are used for controlling translation and mRNA stability
Translation of an mRNA molecule begins at the 5’ end of the
mRNA and proceeds in the 3’ direction.
Polypeptide synthesis begins at the
amino terminus (“NH2)
The sequence of nucleotides in the linear mRNA molecule is read consecutively in groups of
Three. It is read from a fixed starting point: AUGGGGCUCAGCGAC is read
as AUG-GGG-CUC-AGC-GAC–.
Codon
Each group of three consecutive nucleotides in RNA. Each
codon specifies either one amino acid or a ‘stop’ codon to the translation process.
genetic code
The nucleotide sequence of a gene is translated into the amino acid sequence of a protein by rules known as the genetic code. Genetic code is non-overlapping and continuous.
All proteins (ORFs) start with an
AUG codon, which codes for methionine. In prokaryotes, it is a modified methionine called “formyl-methionine.”
Translated is halted at
stop codons
3 nucleotides specificy
1 amino acid (codon)
How to read codons
Three stop codons
UAA, UGA, UAG (you are away, you go away, you are gone)
Specificity of genetic code
a particular codon always codes for the same amino acid
for example: UUA will always code for leucine
Universality of genetic code
its specificity has been conserved from very early stages of evolution
Degeneracy (redundancy) of genetic code
several codons can be used to code for the same AA. For example, leucine is specified by six different codons.
Nonsense mutation
base substitution results in the introduction of a stop (termination) codon (changing a single nucleotide base in the coding region of mRNA)
Silent mutation
base substitution does not change the identity of the incorporated AA (changing a single nucleotide base in the coding region of mRNA)
Missense mutation
base substitution results in an AA change (changing a single nucleotide base in the coding region of mRNA)
The sequence of nucleotides is read consecutively in groups of
three
insertion or deletion of “N” number of bases where “N” is not evenly divisible by 3 alters
the reading frame of the rest of the translated mRNA. This is called a frameshift mutation. This can induce missense and nonsense mutation.
Occasionally, a sequence of three bases that is repeated in tandem will become amplified in number so that
many copies of the triplet occur. If this happens within the coding region of a gene, the protein will contain many extra copies of one AA.
expansion of CAG codon (glutamine) in
exon 1 for huntington protein. Example of tri-nucleotide repeat extension.
The expansion in the coding region results in
abnormally long protein - when cleaved, it produces
toxic fragments that aggregates in neurons, causing
neurodegenerative Huntington disease
Expansion of CGG repeats (arginine)
fragile X syndrome
The abnormal expansion prevents
production of (Fragile X)
FMRP. This loss disrupts nervous system function, leading to signs of fragile X syndrome, the common cause of intellectual disability seen in males.
Expansion of CUG (serine) is seen in
myotonic dystrophy
3 stages of translation
initiation, elongation, termination
mRNAs
-made by RNA polyII
-template
tRNAs
-made by RNApolyIII
-decodes triple codonsAm
Aminoacyl-tRNA synthesase
attach appropriate amino acids to tRNAs with specific anticodons
Ribosomes
site of translation
GTP
GTP is used as the energy requiring steps in translation
ATP
used to charge tRNAs
tRNA
decodes codons
tRNA decodes by using
base pairing between the codon on the mRNA and the anti-codon on the tRNA
Codon and anticodon pairing reflects
complementary (antiparallel) binding
At least one specific type of tRNAs is required for
each amino acid (in humans, there are more than > 50 tRNAs)
all tRNAs have
similar structures
each tRNA has an attachment site for a specific _______ ______ at what end?
specific amino acid, at its 3’ end (AA is attached at the 3’-OH end of the A nucleotide in the -CCA sequence)
An enzyme is required for attachment of AA to their corresponding
tRNA
aminoacyl-tRNA synthetases are responsible for
adding correct AA to their appropriate tRNAs
There are ____ different aminoacyl-tRNA synthesases
20, one for each AA. This specificity contributes to the high fidelity of the translation process.
Aminoacyl-tRNA synthetase enzyme catalyzes a
two step reaction that results in the covalent attachment of an AA to the 3’ end of its corresponding tRNA
1st step of aminoacyl-tRNA synthetase
coupling of AA to AMP to form aminoacyl-AMP
2nd step of aminoacyl-tRNA synthetase
enzyme transfers AA from aminoacyl-AMP to tRNA to form aminoacyl-tRNA (aa-tRNA). aa-tRNA and AMP are released from the enzyme. The overall reaction requires ATP, which is cleaved to AMP and PPi (inorganic pyrophosphate).
aminoacyl-tRNA synthetase has
proofreading and editing activity that can remove an incorrect AA from the enzyme or the tRNA molecule
Aminoacyl-tRNA synthetase steps
- The AA and ATP bind to synthetase
- The AA is attached covalently to AMP, releasing PPi
- Coupling of AA to AMP to form aminoacyl-AMP
- The enzyme binds the appropriate tRNA, and the AA is transferred to the 3’ end of the tRNA. The tRNA is now said to be charged, “charged tRNA” has AA attached to it. An aminoacyl tRNA is a charged tRNA. This has a high energy bond that will help drive the peptide bond formation forward.
Ribosomes are sites of
protein synthesis
mRNAs are translated by
ribosomes
Ribosomes are multi-subunit…
rRNA-containing protein complexes
Two subunits
large and small
Large subunit
catalyzes peptide bond formation.
Small subunit
is a decoding center,
i.e., binds mRNA and determines the
accuracy by insuring correct base-pairing between codon and
anticodon.
A ribosome has three tRNA binding sites
- A (aminoacyl) site: a charged tRNA is presented to the mRNA to be translated
- P (peptidyl) site: the growing peptide chain is attached as a peptidyl-tRNA complex
- E (exit) site: contains empty tRNAs
(Each of the sites extends over both subunits)
In eukaryotic cells, the ribosomes are either
free in the cytosol or in association
with the rough endoplasmic reticulum (RER).
After mRNA processing and splicing are complete, the mature mRNAs are
exported from the nucleus of the cell into the cytoplasm
in the cytoplasm, polypeptides can be synthesized by
free ribosomes in the cytosol or by ribosomes bound to the rough endoplasmic reticulum (RER).
Cytosolic ribosomes
synthesize proteins that are required in the cytosol itself or destined for the nucleus, mitochondria, or peroxisomes.
RER-associated ribosomes
synthesize proteins that are to be exported from the cell, incorporated into membranes, or imported into lysosomes
In eukaryotes, the transcription process must
end before the
translation process can start - described as “uncoupled transcription & translation”
Prokaryotes have “coupled
transcription & translation” process.
For both prokaryotic and eukaryotic, translation factor proteins are required for
translation, and these fall into one of three classifications:
-Initiation factors (IF)
-Elongation factors (EFs)
-Release factors (RFs)
Eukaryotic translation factors are distinguished from prokaryotic by an
“e”
prefix (eIF1, eEF2, eRF1, etc.)
Prokaryotes utilize
Shine-Dalgarno sequence for translation initiation.
Shine Delgarno Sequence
sequences are located 6-10 bases upstream of the start codon (AUG) in the 5’UTR region of the mRNA transcript.
The rRNA of the small ribosomal subunit (FYI, 16S) binds to
Shine-Dalgarno sequence and positions the small ribosomal subunit to promote efficient and accurate translation of mRNA.
Sequences 5’end of the mRNA and the 3’ end of the 16S rRNA form
complementary base pairs.
The initiation tRNA (tRNAiMet)
the tRNA that carries the initiating methionine,
binds to the start codon.
In prokaryotes, the initial methionine carries
N-formylated methionine (fMet)
Translation Initiation in Prokaryotes
- Initiation factors (IFs) and GTP bind to the small ribosomal subunit
- mRNA assemble with the small ribosomal subunit (initiator tRNA recognizes the start codon and bind to the small ribosomal subunit. Binding to mRNA is positioned around the Shine-Delgarno sequence of mRNA.
- Large ribosomal subunit joins the small initiation complex.
- GTP is hydrolyzed and IFs are released.
Now, a functional ribosome is formed with a charged tRNAimet in the P site. The A site is empty.
tRNAiMet is always deposited into the P site.
Translation initiation in eukaryotes occurs in the
nucleus, and translation occurs in the cytoplasm –? referred to as compartmentalization of processes
Eukaryotes mRNA do not have
Shine-Delgarno sequences
Once in the cytoplasm, 5’ cap of mRNA is bound by small
ribosomal subunit along with eIF4G and eIF4E. The subunit moves from the 5’ to 3’ direction along the mRNA until it encounters the initiator AUG.
Proteins that bind to the 5’ cap associates with proteins that
bind to the poly-A-tail –> for efficiency of re-initiation of translation
Translation initiation in eukaryotes (4 steps)
- Eukaryotic initiation begins with the charged initiator tRNA (tRNAiMet) binding to GTP bound eIF2
- eIF2/GTP/tRNA complex binds to the small ribosomal subunit
- Meanwhile, eIF4E/4G bind to the 5’ cap end of the mRNA
- The small ribosomal subunit complex binds to the mRNA/eIF4
