frontiers-in-genetics.

org

mRNA tRNA rRNA proteins ribosomes

Translation
Lecture 13 Fall 2013
Chapter 8

Learning Goals for Translation

Translation
Describe the process of translation; focusing on the unique aspects of bacterial transcription: initiation, operons Know the roles of mRNA, tRNA, rRNA Be able to accurately convert a fragment of DNA into RNA and into protein Compare/contrast replication/transcription/translation (start/stop signals, template, product, direction of synthesis) Name the players in translation and their functions Understand coupled transcription/translation

TP Question 23

Transcription Products

Translation
Players
ribosomes (rRNA and ribosomal proteins) tRNA mRNA other factors

Need to convert sequence of bases into sequence of amino acids

Crick proposed adapter molecule:
amino acid at one end RNA at other end Name?
5 animation from book: http://www.wwnorton.com/college/biology/mbio/animations/main.asp?chno=ch08a01

 convert the language of RNA into that of proteins tRNAs are shaped like a clover leaf (in 2-D) and a boomerang (in 3-D). A tRNA molecule has two functional regions: - Anticodon: Hydrogen bonds with the mRNA codon specifying an amino acid - 3 (acceptor) end: Binds the amino acid

tRNA
5 on left

ATP, enzymes binds a.a.

binds codon

Figure 8.15

The charging of tRNAs is carried out by a set of enzymes called aminoacyltRNA synthetases.

rRNA
3 sizes: 5S, 16S, and 23S

Ribosomes
50S

programmable machines

total: 70S
30S

composed of two subunits, each of which includes rRNA and proteins. In prokaryotes, the subunits are 30S and 50S and combine to 8 form the 70S ribosome.

Ribosomes

3 binding sites, A, E and P

A receives new tRNA P holds growing peptide chain E is exit site for old tRNA Process continues until no tRNA available (stop codon), then growing peptide released
9

Translation of RNA to Protein

Animation: Protein Synthesis

How do we know which codons on mRNA will match with which amino acids?

Eukaryotic http://www.youtube.com/watch?v=5bLEDd-PSTQ

10

The Genetic Code

nucleotide triplets = codons 64 codons: - 61 specify amino acids. Include the start codon - 3 are stop codons. The code is degenerate or redundant. - Multiple codons can encode same amino acid. The code operates universally across species.

Figure 8.11

What amino acids does this mRNA fragment encode? 5 GACAUGUGA 3

1. 2. 3. 4. 5.

aspartic acid-methionine-stop serine-valine-glutamine leucine-tyrosine-threonine serine-histidine-valine valine-alanine-glutamic acid

12

What if we switched the 5 and 3 ends? 3 GACAUGUGA 5

1. 2. 3. 4. 5.

aspartic acid-methionine-stop serine-valine-glutamine leucine-tyrosine-threonine serine-histidine-valine valine-alanine-glutamic acid

13

Solve at Home
The strand below is the template strand of DNA. Transcribe and translate it. 3 GACATGTGA 5

1. 2. 3. 4. 5.

aspartic acid-methionine-stop serine-valine-glutamine leucine-tyrosine-threonine serine-histidine-valine valine-alanine-glutamic acid

14

Translation of RNA to Protein

Polypeptide synthesis occurs in 3 phases: 1) Initiation: which brings the two ribosomal
subunits together, placing the first amino acid in position 2) Elongation: which sequentially adds amino acids as directed by mRNA transcript 3) Termination: which releases the completed protein and recycles ribosomal subunits

Each phase requires a number of protein factors and energy in the form of GTP.

Translation Initiation
In bacteria, how do ribosomes know where to start? (there is no CAP at the 5 end of mRNA and no poly A tail at the 3 end)
Ribosome binding site (Shine Dalgarno site) on mRNA Different than eukaryotes Ribosomes bind and move down mRNA to start codon, AUG (about 6 bases)

How do ribosomes know where to end?

Stop codon (no tRNA to match)

16

Translation Where is RBS? Initiation

IF (Initiation factor) 3 along with 30S recognize binding site
IF2 + initiator tRNA bind start codon IF3 is released and IF1 binds 50S binds, GTP hydrolysis and releases IF1 and IF2

Figure 8.23
17

Translation Elongation
ribosomes move 5 3 on mRNA tRNA can enter A site only if: 1. it is charged 2. other factors present 3. its anticodon matches the codon
EF-Tu (Elongation Factor) GTP binds to tRNA and guides to A site P & A are bound by petidyltransferase aa from P is transfer to tRNA in A EF-G-GTP binds ribosome advancing 50S 1 codon (A translocate into P) 30S advances, pushes uncharged tRNA out in E

18

Figure 8.24

Translation Termination
RF (release factor)

Figure 8.27

Translation cont.
Energy inputs
Need 1 ATP to attach amino acid to tRNA Need GTP to bind tRNA Need GTP to translocate down mRNA

20

Antibiotics that Affect Translation

Streptomycin: Inhibits 70S ribosome formation Tetracycline: Inhibits aminoacyl-tRNA binding to the A site Chloramphenicol: Inhibits peptidyltransferase Puromycin: Triggers peptidyltransferase prematurely Erythromycin: Causes abortive translocation Fusidic acid: Prevents translocation

TP Question 24

22

Protein Modification
Protein structure may be modified after translation: - N-formyl group may be removed by methionine deformylase. - The entire methionine may be removed by methionyl aminopeptidase. - Acetyl groups or AMP can be attached. - Proteolytic cleavages may activate or inactivate a protein.

Protein Folding
Folding of many proteins requires assistance from other proteins called chaperones: - GroEL and GroES chaperones - Form stacked ring with a hollow center - The protein fits inside the open hole. - DnaK chaperones - Do not form rings - Clamp down on a polypeptide to assist folding
Figure 8.32

Protein Secretion
Proteins destined for the bacterial cell membrane or envelope regions require special export systems. tagged with hydrophobic N-terminal signal sequences of 1530 amino acids. - These sequences are bound by the signal recognition particle (SRP).

Protein Degradation

Many proteins contain degradat. signals called degrons. Proteasomes are protein-degrading machines found in eukaryotes and archaea. Bacteria contain ATP-dependent proteases, such as Lon and ClpP.