The Building of Proteins from a Nucleic Acid Template 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 G T C G G A T C C C T A T C

A 2-step Process Transcription DNA RNA 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.

Transcription 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 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.

In Eukaryotic cells, things get a little more complex: RNA Processing In Eukaryotic cells, things get a little more complex: Several types of RNA polymerase are present – each with a slightly different function – I, II, and III. 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 3’ UTR 5’ 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). 5’cap A A U A A A Poly-A tail

5’ Cap and Poly-A Tail These 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. Introns 5’cap A A U A A A Poly-A tail 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). 5’cap A A U A A A Poly-A tail A A U A A A 5’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 rRNA = Ribosomal 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). 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 Structure Of 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: Translation: 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