Protein Synthesis

The Building of Proteins from a Nucleic Acid Template

DNA

• Provides the Template.• Information contained in the SEQUENCE

of N-bases found along the DNA molecule.

• Transferring that information into an Amino Acid SEQUENCE is the trick.

A G C C T A G G G A T A GT C G G A T C C C T A T C

A 2-step Process

1. Transcription

DNA RNA

2. Translation

RNA Protein

Transcription• Production of RNA.

• RNA = Ribonucleic Acid.

– Ribose Sugar.– Single Stranded Molecule.– Uracil instead of Thymine.

Transcription• RNA Polymerase

• Attaches at a Promoter – a sequence of the DNA that indicates where a gene starts.

• With the help of Transcription Factors.

• A “window” in the DNA is opened – ultimately, a gene.

• RNA Nucleotides are added (from 5’ to 3’) in accordance with the DNA parent template (which is read from 3’ to 5’).

Transcription

Transcription

• Complimentary Base Pairing.

• DNA RNA

G - C

C - G

T - A

A - U

Transcription

• Elongation of a single stranded RNA molecule.

• The DNA window closes behind.

Transcription• Termination – a sequence of bases is

reached signaling RNA Polymerase to release.

• The “gene” closes

• The RNA transcript is released.

RNA Processing

In Eukaryotic cells, things get a little more complex:

1. Several types of RNA polymerase are present – each with a slightly different function – I, II, and III.

2. A larger mRNA is transcribed…

RNA Processing

• During Transcription, RNA polymerase II transcribes MORE than just the protein-encoding part of the gene.

• Untranslated Regions (UTRs) are transcribed at the 5’ and 3’ ends.

– The 3’ UTR is called a Polyadenylation signal.

A A U A A A

5’ UTR 3’ UTR

RNA Processing• AFTER Transcription is terminated,

additional alterations are made to the ends of this “pre-mRNA” strand.

• A 5’ cap at the leading (5’) end (composed of a Guanine-like nucleotide).

• A poly-A tail at the trailing (3’) end (composed of many Adenine nucleotides).

A A U A A A5’cap Poly-A tail

5’ Cap and Poly-A TailThese END pieces serve several functions:

1. Help “ferry” the eventual mRNA transcript out of the nucleus.

2. Protect the protein-encoding area from degradation.

3. Help attach the proper end (5’) of the transcript to the place where Translation will take place – the Ribosome.

Further RNA Processing

• RNA splicing – removes portions of the transcript.

• Removed segments are called INTRONS.

• The remaining coding segments are called EXONS.

A A U A A A5’cap Poly-A tail

Introns

Exons

Further RNA Processing

• INTRONS are recognized by snRNPs…– “small nuclear

RiboNucleoProteins”.– snRNPs cut out

INTRONS and join the adjacent EXONS.

– This occurs in the nucleus.

Further RNA Processing

• The resulting molecule is the RNA transcript proper (no longer the pre-mRNA).

A A U A A A5’cap Poly-A tail

A A U A A A5’cap Poly-A tail

Why?

• Introns may be vestigial.

• Introns may have regulatory roles.

• Alternative RNA splicing – more than one polypeptide from a single gene.

• More chances of unique Exon combinations (taking into account “crossing over” during meiosis).– (Cross-overs at introns won’t disrupt exons).

3 Types of RNA

• mRNA = Messenger RNA– The “blueprint”

• rRNA = Ribosomal RNA– The “workbench”

• tRNA = Transfer RNA– The “truck”

mRNA – messenger RNA

• The “message” – the blueprint for the production of a polypeptide – a protein.

• But there’s only 4 N-bases, and 20 amino acids!

• What’s the code?

The Genetic Code• Triplet Base Code.

• Every 3-letter word in the RNA Transcript is a CODON.

The Genetic Code

• Each CODON codes for a single Amino Acid.

The Genetic Code

rRNA – Ribosomal RNA

• The location for protein synthesis – the workbench on which a polypeptide is built.

• rRNA makes up a RIBOSOME.

• Ribosomes have 2 sub-units.

Large subunit

Small subunit

rRNA – Ribosomal RNA

• The Large subunit has several binding sites:

tRNA – Transfer RNA

• The “transfer-er” – the truck that brings individual Amino Acids to the workbench for the production of a polypeptide – a protein.

• With a driver – the Anticodon.

tRNA – Transfer RNA

• Each tRNA carries a specific Amino Acid to the Ribosome.

• It “knows” when to drop this Amino Acid off by…

• COMPLIMENTARY BASE PAIRING of its Anticodon with the corresponding Codon found on the mRNA.

tRNA – Transfer RNA

• Aminoacyl-tRNA synthetase = the enzyme responsible for hooking Amino Acids to tRNAs.

• Aminoacyl tRNA = truck with cargo.

Translation• Translating the RNA

sequence data into Protein sequence data.

• N-bases Amino Acids

• In the cytoplasm of Eukaryotic cells.

Translation• Initiation - All 3 types of RNA come together.

• This requires some energy (provided by Guanosine Triphosphate -- GTP).

Translation• Elongation – Amino Acids are added with

the help of elongation factors (proteins).

1. Codon meets Anticodon at the ‘A’ binding site.

2. A Peptide Bond Forms between adjacent Amino Acids

Translation

• Then, a shift in the Ribosome (a “Translocation”) moves the empty tRNA to the exit (E-site), opening the A-site for the next tRNA.

Translation

• Termination…

• Continued elongation until a “STOP” codon is reached (UAG, UAA, or UGA).

• A release factor (protein) binds to this codon, effectively releasing the polypeptide.

• The mRNA, tRNA, and rRNA separate.

Protein Synthesis

Primary StructureOf the Protein

Protein Synthesis• In response to a need for a particular

protein – need it NOW.

• The same gene can be transcribed by several RNA polymerase molecules simultaneously…allowing faster protein production.

• Polyribosomes – strings of Ribosomes translate a single mRNA simultaneously – allowing faster protein production.

Polyribosomes

Bound and Free Ribosomes• Free-floating Ribosomes are the sites for

free-floating protein production.

• Ribosomes bound to membranes are the sites for membrane-bound and secretory proteins.

Review – Fig. 17.26

Point Mutations• Point Mutations occur at single nucleotides

along DNA’s template strand.

• Substitutions – the replacement of a pair of nucleotides at a particular location.

• Silent Mutations.• Not-So-Silent Mutations.

– Missense– Nonsense

Substitution Mutations

• Original Sequence:

• Silent Mutation:

• Missense Mutation:

• Nonsense Mutation:

Point Mutations

• Insertions & Deletions – adding or removing bases along the sequence.

– Results in a FRAME SHIFT because all other codons “downstream” are affected.

– Can reverse if there are 3 insertions or 3 deletions.

Insertions & Deletions• Original Sequence:

• Insertion/nonsense:

• Deletion/missense:

• 3-Nucleotide Insertion/Deletion (missense):

Assignment:

• Review chapters 16 & 17!Transcription:http://www.ncc.gmu.edu/dna/mRNAanim.htm

http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html

Translation:http://www.ncc.gmu.edu/dna/ANIMPROT.htm

http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a3.html

Overall: http://www.johnkyrk.com/DNAtranslation.html