Lecture 13 Fall 2013

Lecture 13Fall 2013

Translationfrontiers-in-genetics.org

mRNAtRNArRNAproteinsribosomes

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

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3

TP Question 23

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Transcription Products

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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?

animation from book:http://www.wwnorton.com/college/biology/mbio/animations/main.asp?chno=ch08a01

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tRNA

binds a.a.

binds codon

ATP, enzymes

5’ on left

• 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

• The charging of tRNAs is carried out by a set of enzymes called aminoacyl-tRNA synthetases.

Figure 8.15

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rRNA3 sizes: 5S, 16S, and 23S

Ribosomesprogrammable machines

50S

30S

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

total: 70S

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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

Eukaryotic http://www.youtube.com/watch?v=5bLEDd-PSTQ 10

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

• Animation: Protein Synthesis

Translation of RNA to Protein

Figure 8.11

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.

The Genetic Code

What amino acids does this mRNA

fragment encode?

5’ GACAUGUGA 3’

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1. aspartic acid-methionine-stop

2. serine-valine-glutamine

3. leucine-tyrosine-threonine

4. serine-histidine-valine

5. valine-alanine-glutamic acid

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

3’ GACAUGUGA 5’

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1. aspartic acid-methionine-stop

2. serine-valine-glutamine

3. leucine-tyrosine-threonine

4. serine-histidine-valine

5. valine-alanine-glutamic acid

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Solve at Home

The strand below is the template strand of DNA. Transcribe and

translate it.

3’ GACATGTGA 5’

1. aspartic acid-methionine-stop

2. serine-valine-glutamine

3. leucine-tyrosine-threonine

4. serine-histidine-valine

5. valine-alanine-glutamic acid

• 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 of RNA to Protein

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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)

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Translation Initiation

Where is RBS?

Figure 8.23

IF (Initiation factor) 3 along with 30S recognize binding site

IF2 + initiator tRNA bind start codonIF3 is released and IF1 binds50S binds, GTP hydrolysis and releases IF1 and IF2

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Translation Elongationribosomes move 5’ 3’ on mRNAtRNA can enter A site only if: 1. it is “charged” 2. other factors present3. its anticodon matches the codon

Figure 8.24

EF-Tu (Elongation Factor) GTP binds to tRNA and guides to A siteP & A are bound by petidyltransferaseaa from P is transfer to tRNA in AEF-G-GTP binds ribosome advancing 50S 1 codon (A translocate into P)30S advances, pushes uncharged tRNA out in E

Translation Termination

Figure 8.27

RF (release factor)

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Translation cont.

• Energy inputs– Need 1 ATP to attach amino acid to tRNA– Need GTP to bind tRNA– Need GTP to translocate down mRNA

• 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

Antibiotics that Affect Translation

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TP Question 24

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 foldingFigure 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 15–30 amino acids.• - These sequences are bound by the signal

recognition particle (SRP).•

Protein DegradationMany 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.