Transcript Translation

TRANSLATION: information transfer from RNA to protein
the nucleotide sequence of the mRNA strand is
translated into an amino acid sequence.
•This is accomplished by tRNA and ribosomes.
•The amino acid sequence is encoded in the nucleotide sequence
•mRNA strands usually have an AUG sequence which means start and end
with UAA, UAG, or UGA (stop).
•AUG always means methionine, it also means START.
•As a consequence, all polypeptides begin with methionine, at least initially.
The initial methionine is trimmed off in most polypeptides later.
•There are three stop codons, which are not associated with amino acids.
They cause the polypeptide to be released by the ribosome.
•the reading direction is 5’ to 3’.
•Note also the importance of reading frame. It is essential that the ribosome
begin reading at exactly the right position in the nucleotide sequence in
order to create the desired protein.
The following sequence of DNA can be read in six reading frames.
Three in the forward and three in the reverse direction.
The three reading frames in the forward direction are shown with the translated amino
acids below each DNA seqeunce.
Frame 1 starts with the "a"
Frame 2 with the "t"
Frame 3 with the "g".
Stop codons are indicated by an "*"
The longest ORF is in Frame 1.
5'
3'
atgcccaagctgaatagcgtagaggggttttcatcatttgaggacgatgtataa
1 atg ccc aag ctg aat agc gta gag ggg ttt tca tca ttt gag gac gat gta taa
M P K L N S V E G F S S F E D D V *
2 tgc cca agc tga ata gcg tag agg ggt ttt cat cat ttg agg acg atg tat
C P S * I A * R G F H H L R T M Y
3 gcc caa gct gaa tag cgt aga ggg gtt ttc atc att tga gga cga tgt ata
A Q A E * R R G V F I I * G R C I
Amino Acid
SLC
DNA codons
Isoleucine
I
ATT, ATC, ATA
Leucine
L
CTT, CTC, CTA, CTG, TTA, TTG
Valine
V
GTT, GTC, GTA, GTG
Phenylalanine
F
TTT, TTC
Methionine
M
ATG
Cysteine
C
TGT, TGC
Alanine
A
GCT, GCC, GCA, GCG
Glycine
G
GGT, GGC, GGA, GGG
Proline
P
CCT, CCC, CCA, CCG
Threonine
T
ACT, ACC, ACA, ACG
Serine
S
TCT, TCC, TCA, TCG, AGT, AGC
Tyrosine
Y
TAT, TAC
Tryptophan
W
TGG
Glutamine
Q
CAA, CAG
Asparagine
N
AAT, AAC
Histidine
H
CAT, CAC
Glutamic acid
E
GAA, GAG
Aspartic acid
D
GAT, GAC
Lysine
K
AAA, AAG
Ribosomes
• Ribosomes are the organelle (in all cells) where
proteins are synthesized.
• They consist of two-thirds rRNA and one-third
protein.
• Ribosomes consist of a small (in E. coli , 30S) and
larger (50S) subunits. The length of rRNA differs in
each.
• The 30S unit has 16S rRNA and 21 different proteins.
• The 50S subunit consists of 5S and 23S rRNA and 34
different proteins.
• The smaller subunit has a binding site for the mRNA.
The larger subunit has two binding sites for tRNA.
tRNA
• Transfer RNA (tRNA) is basically cloverleaf-shaped.
• tRNA carries the proper amino acid to the ribosome
when the codons call for them.
• At the top of the large loop are three bases, the
anticodon, which is the complement of the codon.
• There are 61 different tRNAs, each having a different
binding site for the amino acid and a different
anticodon.
• For the codon UUU, the complementary anticodon is
AAA.
• Amino acid linkage to the proper tRNA is controlled
by the aminoacyl-tRNA synthetases.
• Energy for binding the amino acid to tRNA comes
from ATP conversion to adenosine monophosphate
(AMP).
Two models of tRNA.
Translation
• Polymerization process, consisting of
initiation, elongation and termination.
• process of converting the mRNA codon
sequences into an amino acid sequence.
• The initiator codon (AUG) codes for the
amino acid N-formylmethionine (f-Met) in
proks. No transcription occurs without the
AUG codon. f-Met is always the first amino
acid in a polypeptide chain, although
frequently it is removed after translation.
• Small ribosome unit binds IF1 and 2 then in
the presence of GTP binds fmet tRNA.
Initiation
3 IF factors
IF1 and 3 cause ribosome to dissociate into 50S and 30s
They bind to 30S
IF2 binds to incoming fMet tRNA together with GTP
The incoming mRNA binds to the 30S unit.
It is lined up correctly by the Shine Dalgarno sequence in the mRNA (10) which is complimentary to a sequence of rRNA
The fmet tRNA then binds to the 30s ribosome unit where the IF 1 and
3 and mRNA are already bound
This is the initiation complex
The 50S ribosome unit then binds to this intiation complex
The 50S unit has 3 sites, P (peptidyl), A (amino acyl) and E (exit).
When the initiation complex and the 50S come together the fMet aligns
with the Psite.
The anticodon end contacts the 30S and the acceptor the 50S
Elongation
• P site is initially occupied by fmet in the
initiation complex.
• The second amino acid binds at the A site.
• The code on the tRNA binds to the
complimentary code on the messenger RNA
• GTP and 2 elongation factors( EfTu and EFT)
guide the amino acyl tRNA in and align the
codon.
• GTP is hydrolysed and EFTu dissociates
• EFT is involved with regeneration of EFTUGTP complex.
• New tRNAs bring their amino acids to the
open binding site on the ribosome/mRNA
complex
• a peptide bond is formed between the amino
acids, catalysed by peptidyl transferase
• Uncharged tRNA moves from P to E sites
• peptidyl tRNA moves from A to P
• The complex then shifts along the mRNA to
the next triplet, opening the A site.
• The new tRNA enters at the A site.
Regeneration of
EF-Tu-GTP
by Tu-Ts
exchange.
A schematic view of functional regions of the ribosome.
Termination
• When the codon in the A site is a termination
codon,(UAA, UAG, UGA) a releasing factor
binds to the site, stopping translation and
releasing the ribosomal complex and mRNA.
• 3 different release factors, RF1,2 and 3
• RF not only block A site but affect the bond
between the tRNA and the growing chain
which is broken releasing the peptide
• Often many ribosomes will read the same
message
• a structure known as a polysome forms
The 70S ribosome is now unstable in the presence
of a protein called ribosome recycling factors, as
well as the initiation factors IF3 and IF1.
Consequently, the 70S ribosome dissociates to
50S and 30S subunits and is ready for another
round of translation.
When the ribosomal subunits separate, the 30S
subunit may not always dissociate from its
mRNA.
For example, in polycistronic messages (more
than one protein coded on an mRNA), the 30S
subunit may simply slide along the mRNA until
the next Shine-Dalgarno sequence and initiation
codon are encountered and begin a new round
of translation.