Class I tRNA

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Transcript Class I tRNA

Prokaryotic Translation
Three stages
Initiation: binding of
ribosome (containing rRNAs
and proteins) and aminoacyl
tRNA to mRNA.
Elongation: addition of one
aa at a time to the growing
polypeptide chain.
Termination: release of
finished polypeptide from
tRNA and dissociation of
ribosome from mRNA.
Preinitiation:
tRNA charging;
Dissociation of ribosome
Structure of tRNA
D
Structure of
aminoacyl-tRNA
Length: 76 (74-95) residues.
Extra arm (variable loop):
Class I tRNA: 3-5 bases
Class II tRNA: 13-21 bases;
~5 bases in the stem.
Additional types of base pairing:
G·U, G·Y, A·Y.
Less stable
Positions:
Invariant: maintain 2O structure
Semiinvariant (other Pu or Py)
Name of tRNA:
Tyr
tRNA1
Tyr
tRNA2
Name of charged tRNA: Tyr-tRNA
A
C
C
I
tRNA are
processed from
longer precursors
Modified
nucleosides
found in
tRNA
The bases are
modified (>50
different types)
after transcription
by specific tRNAmodifying
enzymes to affect
the efficiency of
charging and
pairing properties.
Y
3-D Structure of tRNA
RNA-RNA double
helices (11 bp/turn)
Tertiary structure of tRNA is
created by H-bonding:
Secondary H-bonds;
Tertiary H-bonds (formed
between unpaired invariant
and semiinvariant bases)
tRNA charging
Activated amino acid
At least 20 synthetases exist, one for
each amino acid. Isoaccepting tRNAs
are recognized by the same synthetase.
tRNA charging
Recognition depends on an interaction between a few
points of contact in tRNA, mostly at the acceptor stem
and anticodon, and a few amino acids constituting the
active site in the enzyme.
Binding of tRNA synthetase with tRNA
Two classes of tRNA synthetase:
Class I tRNA synthetases
Aminoacylate the 2’-OH group of the terminal A of
the tRNA.
Approach the tRNA from the D-loop and acceptor
stem minor groove side.
Class II tRNA synthetases
Aminoacylate the 3’-OH group of the terminal A of
the tRNA.
Approach the tRNA from the variable arm and
acceptor stem major groove side (the opposite side
of tRNA that contacts the class I enzyme).
tRNA Synthetase
Location of
varies
Each class contains about 10 enzymes
Binding of tRNA synthetase with tRNA
Class I
Class II
Recognition of correct
tRNA by tRNA synthetase
is achieved by two steps:
Association
Aminoacylation
Recognition of correct
amino acid by tRNA
synthetase is also
achieved by two steps
(every synthetase
undergoes
proofreading at either
stage), which occurs
only in the presence
of cognate tRNA.
Ile-tRNA synthetase
has two active sites
for sieving the
cognate amino acid
Synthetic site:
activation of amino acid
Editing site:
hydrolysis of incorrrect
aminoacyl-tRNA
Accuracy of charging tRNAIle by its synthetase
depends on error control by two steps
Meaning of tRNA is determined by its anticodon alone
Structure of Ribosome
r-
30S
Remove Mg 2+
70S
50S
Arrangement of proteins and 16S rRNA in S30 subunit
Central domain
RNA is concentrated at the interface with the 50S subunit.
Both 30S and 50S subunits are self-assembled in vitro. In
30S subunit, S4 and S8 bind to 16S rRNA first, other proteins
then join sequentially and cooperatively.
16S rRNA
Secondary structure
of 16S rRNA and
interaction of this
RNA with proteins
and tRNA were
studied by primer
extension and
crosslinking, and
other techniques.
Features of rRNAs
rRNAs have considerable 2o structure (This is analyzed by comparing
the sequences of corresponding rRNAs in related organisms).
About 2% of the residues in rRNAs are methylated, which may be
important for ribosomal function.
Interaction of rRNA with some ribosomal protein induce conformational
change of rRNA so that it can interact with another protein.
rRNA interacts with mRNA or tRNA at each stage of translation. The
proteins are necessary only to maintain the rRNA in a structure in
which rRNA can perform the catalytic function. Conformation of rRNAs
is flexible during protein synthesis.
The 3’ terminus of 16S rRNA pairs with the SD sequence of mRNA at
initiation.
rRNA contacts the tRNA at parts of the structure that are universally
conserved.
Translation
initiation
1.
Dissociation of ribosome.
2.
Binding of IF-3 to 30S subunit to
prevent reassociation of ribosome.
3.
Binding of IF-1 and IF-2 (with GTP)
alongside IF-3.
4.
Binding of mRNA and fMet-tRNAfMet
to form 30S initiation complex.
5.
Binding of 50S subunit with loss of
IF-1 and IF-3.
6.
Dissociation of IF-2 with hydrolysis
of GTP to form 70S initiation
complex.
Recycle of
ribosome and
initiation
factors
Ribosomes bind to mRNA at a special sequence
Ribosomal protein
S12 and 16S rRNA,
are responsible for
recognition of the
SD sequence. A
conserved sequence
near the 3’-end of
16S rRNA pairs with
the SD sequence.
mRNA sequence protected by ribosome
(about 30 nt)
Ribosomal binding to the SD sequence provides a means for
controlling gene expression.
IF-3 is the primary factor for mRNA binding to ribosome. IF-1 and IF-2,
which bind near IF-3, assist assembly of 30S initiation complex.
fMet-tRNAf as initiator tRNA
This initiator tRNA recognizes codons AUG (90%), GUG (8%),
or UUG (1%) that lies within a ribosome-binding site.
Met
tRNAf is different
Met
from tRNAm ; the
former goes to the
first AUG codon and
the methionine of the
later can not be
formylated.
Formation of fMet-tRNAf
Formylation is not strictly
necessary for the
initiator tRNA to function
in initiation.
It is the tRNA part of
fMet-tRNAf that makes it
the initiating aminoacyltRNA.
IF-2 ensures only the
initiator tRNA goes to
the P-site at initiation.
Features of
Met
fMet-tRNAf
Formation of 70S
initiation complex
30S-mRNA complex
Initiator tRNA joins
IF2-GTP joins complex
IF2 is needed to bind fmettRNAf to 30S-mRNA complex
50S joins and all
factors are released
Removal of formyl group
Deformylase
Removal of
methionine
Aminopeptidase
In bacteria and mitochondria, formyl
group, and sometimes the methionine,
is removed during translation.