Molecular Biochemistry Lecture 10

1
Molecular Biochemistry Lecture 10 From RNA to
Protein Translation
Instructor Dr. Susan Baker phone
x66910 email sbaker1_at_lumc.edu Bldg 105, Rm 3929
2
From RNA to Protein Translation Key Concepts
and Learning Objectives

• An mRNA sequence is decoded in sets of three
  nucleotides.
• An mRNA is decoded on ribosomes using tRNAs.
• The nucleotide sequences in mRNA signal where
  to start protein synthesis.
• Quality control mechanisms operate at many
  stages of translation.
• Ribosomal frameshifting allows translation of
  alternative ORFs.
• Inhibitors of prokaryotic protein synthesis are
  useful antibiotics.

3
(No Transcript)
4
1 1 code, only 4 amino acids? A A
methionine 2 1 code, only 12 amino acid? AT
AU methionine\ 31 code, 64 possible amino
acids? ATG AUG methionine But more
combinations than AAs, so code must be redundant
5
mRNA is decoded in sets of 3 nucleotides
6
RNA pol II
tRNAs ribosomes
7
Decoding the mRNA sequence
For every codon (set of 3 nucleotides)
there exists a corresponding anti-codon in a
tRNA. Each tRNA carries a specific amino acid.
8
tRNA structure anticodon loop
9
The third position in the anticodon is the
wobble base
10
tRNA structure unusual bases
11
Some of the unusual nucleotides found in tRNAs
12
tRNA structure Attachment of amino acid
13
Amino acid activation of the tRNA
14
Structure of the aminoacyl-tRNA linkage
15
(No Transcript)
16
(No Transcript)
17
(No Transcript)
18
(No Transcript)
19
From RNA to Protein Translation

• Recap The mRNA is decoded on ribosomes.
• Describe the structure of tRNA and how it matches
  to codons in the mRNA.
• Define tRNA, anticodon, and wobble-bases.
• Describe the process of incorporation of an amino
  acid into a protein.

20
From RNA to Protein Translation

• An mRNA is decoded on ribosomes.

21
(No Transcript)
22
(No Transcript)
23
Ribosomes associated with the endoplasmic
reticulum
24
The ribosome has 4 binding sites
25
A sequence has 3 possible open reading frames
(ORFs)
mRNA 5-CUCAGCGUUACCAU-3
ORF1
ORF2
ORF3
26

• aminoacyl-tRNA
• binds to A site

• tRNA moves over to P site,
• new peptide bond is formed

3) Ejection of spent tRNA
27
Repeat the cycle!
28
Incorrect tRNA is energetically unfavorable and
dissociates
29
Correct tRNA allows for efficient translation
GTP hydrolysis helps to drive the reaction!
30
How do all these pieces fit together and work
efficiently together?? RNAProtein interactions!!
31
The ribosome is 2/3 RNA and 1/3 protein Crystal
structure solved in 2000
32
(No Transcript)
33
The ribosome is a ribozyme with RNA enzymatic
activity
Gray RNA Gold protein
34
Possible reaction mechanism for the peptidyl
transferase activity The RNA helps to catalyze
the reaction!
35
(No Transcript)
36
From RNA to Protein Translation

• The nucleotide sequences in mRNA signal where
  to start protein synthesis.
• Scanning model and role of Kozak consensus
  sequence in initation of translation

37
Kozak scanning model context is important
5-7mGACGUGGCUAUGGAAGCGAUU.
-3
4
Ribosomes enter at the 5 end of the mRNA And
scan down the RNA to look for the first AUG that
is in good context. The nucleotides that
influence initiation are A/G at 3 and G at 4
38
Kozak scanning model context is important
Where will translation initiate??
5-7mGACGAUGGCUAUGGAAGCGAUU.
5-7mGACGUCCCUAUGAAGCAUGGAUU.
39
Note that 5 and 3 ends of the mRNA are brought
together
40
(No Transcript)
41
What if you dont have a 5 end cap?
Example Poliovirus RNA
5
ORF
AAA
VPg
42
What if you dont have a 5 end cap?
Example Poliovirus RNA
IRES
5
ORF
AAA
VPg
IRES internal ribosomal entry site
43
Exploit IRES to make bi-cistronic mRNAs
57mG
ORF1
ORF2
AAA
Protein 1
IRES
57mG
ORF1
AAA
ORF2
Protein 1
Protein 2
44
Shine-Dalgarno sequences are ribosome- binding
sites in procaryotic mRNA
45
Release factors direct the termination of
translation
46
(No Transcript)
47
Structure of eRFI human translation release
factor It looks like a tRNA!
48
From RNA to Protein Translation

• Quality control mechanisms operate at many
  stages of translation.

49
(No Transcript)
50
Example of rescue of a bacterial ribosome stalled
on an incomplete mRNA
51
(No Transcript)
52
Incorporation of seleocysteine into a protein
53
Note suppression of the stop codon
54
From RNA to Protein Translation

• Ribosomal frameshifting allows translation of
  alternative ORFs.

55
Retroviruses (HIV) use frameshifting to express
GAG-POL
56
Ribosomal frameshifting in coronaviruses use of
the RNA pseudoknot
57
The ribosome has been translating the mRNA and
has just reached the slippery sequence (T TTA
AAC). Each of the anticodons on the tRNAs in the
ribosome has three contacts with its
corresponding mRNA codon.   The two tRNAs slipped
back one nucleotide. Now only two out of the
three nucleotides on each tRNA match with the
mRNA, so there are only two contacts between the
mRNA codons and the tRNA anticodons. However,
this is enough to hold the mRNA and tRNA
together.     The ribosome continues translating
and moves on to the next codon. Because of the
frameshift, the codon is CGG and an arginine is
inserted, while without the frameshift the codon
would have been GGG, signifying a glycine.
58
Ribosome encounters RNA pseudoknot that forces
it to slide back one nucleotide
www.imb-jena.de/csc/NANA.html
59
From RNA to Protein Translation

• Inhibitors of prokaryotic protein synthesis are
  useful antibiotics.

60
Table 6-3 Inhibitors of Protein or RNA Synthesis
Inhibitor Specific Effect Acting only on
bacteria Tetracycline blocks binding of
aminoacyl-tRNA to A-site of ribosome Streptomycin
prevents the transition from translation
initiation to elong Chloramphen. Blocks peptidyl
transferase reaction on ribosomes Erythromycin blo
cks translocation reaction on ribosimes
Rifamycin blocks initation of RNA chains by
binding to RNA pol Acting on bacteria and
eucaryotes Puromycin causes premature release of
nascent polypeptide Actino D binds to DNA and
blocks the movement of RNA pol Acting on
eucaryotes but not bacteria Cyclohex. Blocks
traslocation reaction on ribosomes Anisomycin bloc
ks peptidyl transferase reaction on
ribosomes A-amanitin blocks mRNA synthesis by
binding preferentially to RNA pol II
61
From RNA to Protein Translation concepts
discussed today

• An mRNA sequence is decoded in sets of three
  nucleotides.
• An mRNA is decoded on ribosomes.
• The nucleotide sequences in mRNA signal where
  to start protein synthesis.
• Quality control mechanisms operate at many
  stages of translation.
• Ribosomal frameshifting allows translation of
  alternative ORFs.
• Inhibitors of prokaryotic protein synthesis are
  useful antibiotics.