PROTEIN SYNTHESIS

Protein Synthesis: overview  DNA is the code that controls everything in your body In order for DNA to work the code that it contains must be transcribed and translated into proteins In order for DNA to work the code that it contains must be transcribed and translated into proteins  Two hypotheses One gene-one enzyme hypothesis (Beadle and Tatum) One gene-one enzyme hypothesis (Beadle and Tatum) One gene-one polypeptide (protein) hypothesis One gene-one polypeptide (protein) hypothesis

Overview of Protein Synthesis  Transcription: synthesis of RNA under the direction of DNA (mRNA) synthesis of RNA under the direction of DNA (mRNA)  Translation: actual synthesis of a polypeptide under the direction of mRNA actual synthesis of a polypeptide under the direction of mRNA

The Triplet Code  The genetic instructions for a polypeptide chain are ‘written’ in the DNA as a series of 3-nucleotide ‘words’  Each triplet in DNA codes for a specific amino acid in a protein  Codons Are what we call the 3- nucleotides in DNA which code for amino acids Are what we call the 3- nucleotides in DNA which code for amino acids

Process of Transcription  RNA polymerase: pries DNA apart and hooks RNA nucleotides together from the DNA code  Promoter region on DNA: where RNA polymerase attaches and where initiation of mRNA begins  Terminator region: sequence that signals the end of transcription  Transcription unit: stretch of DNA transcribed into an RNA molecule  Structure of mRNA Complimentary to DNA except Thymine is replaced by another base called Uracil Complimentary to DNA except Thymine is replaced by another base called Uracil Ribose sugar in backbone Ribose sugar in backbone

Process of Transcription  Initiation- transcription factors mediate the binding of RNA polymerase to an initiation sequence (TATA box)  Elongation- RNA polymerase continues unwinding DNA and adding nucleotides to the 3’ end- 60 nucleotides per second  Termination- RNA polymerase reaches terminator sequence Prokaryotes stop right away Prokaryotes stop right away Eukaryotes go nucleotides further than termination sequence Eukaryotes go nucleotides further than termination sequence

Figure 17.6 The stages of transcription: initiation, elongation, and termination

RNA PROCESSING  In eukaryotes…  The 5’ end of pre-mRNA is capped with guanine  Poly(A) tail – several adenine added to 3’ end Protects end Protects end Signal for future ribosome attachment Signal for future ribosome attachment Help to get mRNA out of nucleus Help to get mRNA out of nucleus Help prevent degradation Help prevent degradation

Figure 17.8 RNA processing; addition of the 5 cap and poly(A) tail

RNA SPLICING  In eukaryotes…  Large portions of mRNA do not code for parts of a protein Introns – noncoding segments Introns – noncoding segments Exons – coding segments Exons – coding segments  snRNPs (small nuclear ribonucleoproteins) combine with proteins to make spliceosome  Spliceosomes cut at ends of introns and rejoins remaining exons together (recognize special sequences)  Ribozymes – mRNA that catalyzes its own intron removal (not all enzymes are proteins)

Figure 17.9 RNA processing: RNA splicing

Figure The roles of snRNPs and spliceosomes in mRNA splicing

WHY INTRONS?  Split genes can code for different proteins or different regions of same polypeptide  Introns increase the cross over frequency between 2 alleles which increases diversity

Figure Correspondence between exons and protein domains

TRANSLATION

Translation  Message coming from the mRNA is now translated in the cytoplasm into a polypeptide  tRNA is the interpreter that reads the codon from the mRNA through its anti-codon and carries the correct amino acid that matches.  Structure of tRNA About 80 nucleotides long forming and 3 dimensional shape that looks like and L About 80 nucleotides long forming and 3 dimensional shape that looks like and L Each is unique and has its own anti-codon Each is unique and has its own anti-codon Has an amino acid attachment site Has an amino acid attachment site

How are tRNA’s attached to the amino acid?  Through the use of Aminoacyl-tRNA- synthetases Enzyme that joins the correct amino acids with the tRNA through the hydrolysis of ATP Enzyme that joins the correct amino acids with the tRNA through the hydrolysis of ATP Happens freely in the cytoplasm Happens freely in the cytoplasm

Figure An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA

Steps in Translation  Initiation Brings together mRNA, tRNA, and the two sub units of the ribosome Brings together mRNA, tRNA, and the two sub units of the ribosome mRNA begins being read at the start signal and will go in a 5’ to 3’ direction mRNA begins being read at the start signal and will go in a 5’ to 3’ direction mRNA, tRNA, and small ribosomal unit bind first mRNA, tRNA, and small ribosomal unit bind first Then, large ribosomal unit attaches to the tRNA with the tRNA sitting in the P site and the A site is ready for the next tRNA Then, large ribosomal unit attaches to the tRNA with the tRNA sitting in the P site and the A site is ready for the next tRNA

Figure The initiation of translation

Steps in Translation  Elongation mRNA strip is continuously read and as this happens tRNA’s are bringing amino acids and making a polypeptide mRNA strip is continuously read and as this happens tRNA’s are bringing amino acids and making a polypeptide tRNA binds to A site tRNA binds to A site tRNA in P site transfers polypeptide to tRNA in A site tRNA in P site transfers polypeptide to tRNA in A site Free tRNA in P site moves into E site and exits Free tRNA in P site moves into E site and exits tRNA in A site moves to P site and new tRNA comes into A site tRNA in A site moves to P site and new tRNA comes into A site

Figure The elongation cycle of translation

Steps in Translation  Termination Elongation continues until a stop codon is reached at the A site of the ribosome Elongation continues until a stop codon is reached at the A site of the ribosome Protein called release factor binds to the A site Protein called release factor binds to the A site Due to the addition of water to the polypeptide it gets released and the translation unit breaks down Due to the addition of water to the polypeptide it gets released and the translation unit breaks down

Figure The termination of translation

Polyribosomes  Typically a single mRNA strand is used to make many copies of the polypeptides it codes for simultaneously  Many ribosome's can be bonded to the same mRNA strip all at once  Polypeptides with specific destinations Some polypeptides need to leave the cell Some polypeptides need to leave the cell Therefore they are made in bound ribosome's on the ER and other membrane bound organelles for transport Therefore they are made in bound ribosome's on the ER and other membrane bound organelles for transport

Figure Polyribosomes

Figure The signal mechanism for targeting proteins to the ER

MUTATIONS  Mutation – a change in DNA sequence  Point Mutations cause: missense mutations no change in amino acid(s) missense mutations no change in amino acid(s) nonsense mutations changes amino acid and therefore protein nonsense mutations changes amino acid and therefore protein  Two types of Point Mutations Base pair substitutions replacement of nucleotide Base pair substitutions replacement of nucleotide Insertions and Deletions -additions or losses of one or more nucleotides Insertions and Deletions -additions or losses of one or more nucleotides Frameshift mutation - occurs when number of nucleotides inserted or deleted is not 3 or a multiple of 3Frameshift mutation - occurs when number of nucleotides inserted or deleted is not 3 or a multiple of 3  Mutation rate is ~1 nucleotide altered in every 10 10

Figure The molecular basis of sickle-cell disease: a point mutation

Figure Categories and consequences of point mutations: Base-pair insertion or deletion

Figure Categories and consequences of point mutations: Base-pair substitution

MUTAGENS  Physical or chemical agents cause DNA to mutate X-rays X-rays UV light UV light Radiation Radiation Most carcinogens Most carcinogens

A gene is more than just a protein maker.  A gene is a region of DNA whose final product is protein or RNA  Types of RNA made include mRNA, tRNA, rRNA, snRNA, SRP RNA (part of signal recognition particle), snoRNA (small nucleolar RNA helps process pre-rRNA), and siRNA (small interfering RNA) and miRNA (micro RNA) both involved in gene regulation mRNA, tRNA, rRNA, snRNA, SRP RNA (part of signal recognition particle), snoRNA (small nucleolar RNA helps process pre-rRNA), and siRNA (small interfering RNA) and miRNA (micro RNA) both involved in gene regulation

Figure A summary of transcription and translation in a eukaryotic cell