| M W F; 10:10 - 11:00 pm |
|
Douglas W. Smith |
| York 2722 |
|
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
Readings: Brown, 10: 243-248
Outline:
A. Basics of Translation Process:
As with DNA replication and
RNA transcription, one has:
Initiation .. Elongation .. Termination
B. Initiation:
1. Prokaryotic:
a. Process: ... [Brown, Fig 10.14]
Ribosomes exist in cytoplasm as pools of 30S, 50S, and 70S ribosomes
ONLY 30S and 50S ribosomes participate in Initiation of Translation
Three Initiation Factors (IF-1, IF-2, IF-3) are required for Initiation:
An mRNA - 30S ribosome complex; FOLLOWED BY addition of fMet-tRNAf
Sum: fMet-tRNAf 30S ribosome mRNA 50S ribosome
| | IF-3 (IF-1) | |
| |_____________| |
| IF-2 | |
|_____________________| |
| GTP |
|________________________________|
| IF-2, IF-3 leave; GTP hydrolysis
|
fMet-tRNAf - mRNA - 70S ribosome complex
1. IF-3 binds 30S ribosomes, enabling 30S ribosome to bind mRNA and preventing 50S ribosome from binding to 30S ribosome ... This forms the 30S-mRNA-IF3 complex
2. IF-1 binds 30S ribosomes; function probably is to stablize the Initiation Complex
3. IF-2 binds fMet-tRNAf, brings this activated Initiation tRNA to the 30S-mRNA-IF3 Complex; facilitates insertion of the fMet-tRNAf into P site of 30S ribosome; and activates a GTPase activity to facilitate final joining of 50S ribosome to the Initiation complex
IF-2 and IF-3 (and probably IF-1) dissociate when 50S ribosome binds
b. mRNA has two recognition sites: ...[Brown, Fig 10.13]
1. The Shine-Delgarno ribosome binding site AGGAGG, complementary in sequence to a CCUCCU sequence on 16S rRNA on the 30S ribosome ... [Brown, Table 10.4]
2. The translation Start Codon, usually AUG, sometimes GUG
The 30S ribosome binds the Shine-Delgarno site on the mRNA. Thus, in prokaryotes, ribosomes can begin translation internal on an mRNA. This is as needed, since prokaryotic mRNA species are often transcripts of operons of more than one gene, i.e. they are often multi-cistronic transcripts.
c. fMet-tRNAf - A specific aminoacyl-tRNA used in Initiation: ...[Brown, Fig 10.5 B]
The formylation reaction occurs
after tRNAf is charged with Methionine
This reaction uses formyl-tetrahydrofolate as formyl donor
tRNAf differs from
tRNAMet and other tRNAs as follows:
1) CA unpaired bases in Acceptor
Stem => no function in elongation
2) Three G:C pairs in Anticodon
Stem => can enter P site in 30S ribosome
tRNAMet can not be formylated and is used only for internal methionines ...
A deformylase removes the Formyl group during the Termination process; the Met is also sometimes removed ...
2. Eukaryotic: ... [Brown, Fig 10.15]
Eukaryotic (e) Initiation Factors
(eIFs) play similar roles in Eukaryotes ...
but there are more factors ...
Met-tRNAi - 40S ribosome complex; FOLLOWED BY mRNA addition
Sum: Met-tRNAi 40S ribosome mRNA 60S ribosome
| GTP,eIF-2 | eIF-4A,4B| |
|_______________| | |
| eIF-3; ATP | |
|_____________________| |
| eIF-6 |
|______________________|
eIF-5,4C | eIF-2, eIF-3 leave
|
Met-tRNAi - mRNA - 80S ribosome complex
1. The Met-tRNAi - 40S ribosome complex:
Like prokaryotes, a special tRNAi is used for initiation, also charged with a Met ... BUT the Met is NOT formylated ...
eIF-2 plus GTP binds Met-tRNAi in a Ternary Complex; this complex then enters the P site of 40S ribosome
2. Unlike prokaryotes, eukaryotic Initiation Factors (eIFs) bind to 5' end of mRNA, facilitate binding of, and moving to, site of protein translation:
a. CAP Binding Protein (CBP) binds to the 5' Cap of eukaryotic mRNA
b. eIF-4A then unwinds mRNA 2° structure for about 15 bases, and is joined by eIF-4B for further unwinding of the mRNA; ATP hydrolysis provides energy
c. eIF-3 facilitates binding of the Met-tRNAi - 40S ribosome complex to the mRNA
d. Met-tRNAi - 40S migrates along mRNA to AUG start codon .. ATP is probably used for energy; eIF-4A and eIF-4B probably dissociate
e. eIF-6 brings 60S ribosome to Met-tRNAi - 40S - mRNA complex
f. 60S ribosome joins when
eIF-5 causes release of eIF-2 and eIF-3.
eIF-4C is also required for this 40S - 60S joining reaction
g. When joining is complete, all eIF factors dissociate, leaving:
Met-tRNAi - 40S - 60S - mRNA
C. Elongation ... [Brown, Fig 10.16]
1. Prokaryotic:
a. Loading Ribosome with AminoAcyl-tRNA molecules:
Elongation factor EF-Tu
uses energy from GTP to bring Activated
tRNA molecules to the A site of 70S ribosomes whose P site is
already occupied
EF-Tu - GDP is then released; this is the Inactive form of EF-Tu
Tu-Ts: GDP is then
released by Tu; Tu complexes with elongation factor Ts
The Tu-Ts complex remains intact until GTP reactivates Tu, releasing
Ts
70,000 Tu molecules per cell, nearly 1 per tRNA, vs only 10,000 Ts molecules per cell
b. Translocation and Peptide Chain Elongation
Peptidyl transferase catalyzes transfer of aa from tRNA to the polypeptide chain; this reaction may well be catalyzed mainly by 23S rRNA ... [Brown, Research Brief 10.2]
Translocation then occurs: factor EF-G and GTP
20,000 EF-G per cell, about 1 per ribosome; released upon GTP hydrolysis
The tRNA Acceptor stem moves to the new sites first; then Anticodon stem moves ... Translocation
Ribosomes bind EF-Tu and EF-G separately, resulting in a Cyclic, Sequential process
2. Eukaryotic:
Elongation Factor eEF-1
is similar to EF-Tu, and is often called EF-T ...
with GTP, brings AminoAcyl-tRNA to A site
Elongation Factor eEF-2 is similar to EF-G: a GTP-dependent translocase
D. Termination: ... [Brown, Fig 10.17]
Stop Codons (Nonsense Codons):
universally used - Prokaryotes; Eukaryotes
UAG (amber), UAA (ochre), UGA (opal)
1. Releasing Factors
a. Prokaryotes:
RF1: recognizes UAG and UAA; RF2: recognizes UAA
and UGA
only ~600 molecules per cell, 1 per 50 ribosomes
require Peptidyl-tRNA in P site; act at the A site
b. Eukaryotes:
eRF: recognizes all 3 stop codons ... requires GTP for release
Termination Reaction:
Release of completed PolyPeptide from last tRNA; expulsion of tRNA; dissociation of ribosome from mRNA as separate subunits: 30S and 50S, or 40S and 60S
ORF: open reading frame, no stop codons
URF: unidentified reading frame: protein known to be made;
function unknown
If you have problems or comments, send email to Doug
Smith