PROTEIN SYNTHESIS

CENTRAL DOGMA OF MOLECULAR BIOLOGY: DNA is used as the blueprint to direct the production of certain proteins

What is a gene? Gene = a segment of DNA coding for a RNA segment. –These RNA segments will be used to produce a polypeptide (structural or enzymatic protein) Each strand of DNA can contain thousands of genes Each gene has a precise beginning and an end

Gene Expression The DNA nucleotide sequence codes for the order in which amino acids are put together to form proteins Every three nucleotides on the mRNA (codon) codes for a specific amino acid

Genetic Code 20 different amino acids but 64 possible codons –4 3 = 64 –Some redundancy - more then one codon codes for the same amino acid Code is universal in almost all organisms

Transcription Information is transferred from DNA to RNA Occurs in the nucleus

Types of RNA All three types of RNA are transcribed from DNA –Messenger RNA – carries the coded message from the DNA to the ribosome in the cytoplasm –Ribosomal RNA – reads the mRNA –Transfer RNA – transfers the correct amino acid to the ribosome

Overview of Transcription The segment of DNA that contains the gene for a specific protein or RNA that the cell wants to produce will unwind and the complementary RNA strand will be made by incorporation the RNA nucleotides

Stages in Transcription Each gene has a precise beginning known as the promotor region and an end known as the termination sequence

Stages in transcription of RNA Initiation –Transcription factors bind to the promoter region (TATA box) of the DNA –RNA polymerase then initiates transcription by binding to the transcription factor Unwinds the DNA Elongates the RNA segment

Stages in transcription of RNA Elongation –Nucleotides are added in the 5’ to 3’ direction by RNA polymerase –They form temporary hydrogen bonds with the DNA template –As the DNA helix reforms the RNA peels away

Stages in transcription of RNA Termination –At the end of the gene the termination sequence causes transcription to end –Pre-RNA segment dissociates from the DNA Terminator

Post-transcriptional Modifications mRNA 5’ cap – a guanine triphosphate is added –signal for ribosomal attachment in the cytoplasm 3’ poly A tail – polyA polymerase adds ~250 “A” nucleotides to the end –protects RNA from being degraded by nucleases

Post-transcriptional Modifications mRNA Splicing –Exons – coding region –Introns – noncoding region Cleaved out by snRNPs, and exons are spliced together

Post-transcriptional Modifications Pre - mRNA Mature mRNA

Post-transcriptional modifications rRNA rRNA associates with proteins to form two subunits (40s and 60s) Leaves the nucleus and enters the cytoplasm

–Folds into a three dimensional structure (clover shaped) Post-transcriptional Modifications tRNA

Translation Going from the mRNA nucleotide code to amino acid code mRNA is read by a ribosome (rRNA) to determine the sequence of amino acids Occurs in the cytoplasm

Translation Players in Translation –mRNA strand –Ribosomes (rRNA) –tRNAs carrying amino acids –enzymes

rRNA rRNA has a mRNA binding site and three tRNA binding sites –A site (amino-acyl binding site) –P site (peptidyl binding site) –E site

Has an anticodon three base sequence that is complementary to a codon on the mRNA 3’ end of the tRNA contains a binding site for a specific amino acid tRNA

Stages of Translation Initiation –mRNA binds to the 40s ribosome subunit –The initiator tRNA binds to the mRNA start codon (AUG) at the P site on the ribosome –The arrival of the 60s subunit completes the initiator complex

Stages of Translation Elongation –The next tRNA enters at the A site –The enzyme peptidyl transferase forms a peptide bond between the amino acid on the P site and the new amino acid on the A site.

Stages of Translation: Elongation –The ribosome then moves down the mRNA (translocation) –The tRNA that was at the A site is now at the P site and the empty tRNA that was at the P site now exits at the E site

Charging of tRNA Amino acids are floating freely in the cytoplasm The enzyme amino-acyl tRNA synthetase attaches the amino acids to the 3’end of the tRNA Requires ATP

Stages of Translation Termination –Elongation continues until a stop codon on the mRNA is reached (UAA, UAG, UGA) –The polypeptide is then released from the ribosome by a release factor

Polysomes Several ribosomes can simultaneously translate the same mRNA strand to make multiple copies of the same polypeptide

The start methionine is removed by the enzyme aminopeptidase Protein will under go folding or modifications –Cleavage into smaller fragments or joined with other polypeptides –Chemical modifications: addition of carbohydrates or lipids –Transport to its destination Post-translational modifications to the Polypeptide

Bigger Picture: What happens to these proteins once they are made?

Summary

Mutations and their consequences Mutation = a change in the sequence of bases within a gene –Caused by a mistake during DNA replication (rare) –Or due to environmental factors called mutagens Mutations can be somatic or germinal What is the main difference in the outcome of germinal mutation compared to the outcome of somatic mutation?

Types of Mutations Point mutations (substitutions) – change in a single nucleotide –Due to redundancy of the genetic code it may change the amino acid, it may not “wobble” –Silent mutations do not change the protein Normal hemoglobin DNA mRNA Sickle-cell hemoglobin ValGlu Protein

Types of Mutations Frame-shift mutation – caused by insertion or deletion of a nucleotide –Changes the reading frame of the codons, usually results in a non-functional protein LeuAlaHis PheGlyAla

Although mutations are sometimes harmful….. –They are also the source of the rich diversity of genes in the world –They contribute to the process of evolution by natural selection Are all mutations bad?