Cell Structure Function FALL 14 EXAM III Thomas Keller Topic 21 Protein Synthesis and Targeting Transcription and Translation Topic 22 Protein Synthesis and Targeting Endoplasmic Reticulum Topic 23 Protein Synthesis and Targeting Golgi Secretion Viruses and Lysosomes Topic 24 Protein Synthesis and Targeting Vesicle Targeting Topic 25 Protein Synthesis and Targeting Mitochondria Chloroplasts and Nucleus Topic 26 Actin Filaments Topic 27 Actin Filament Architectures Myosins and Muscle Structures Topic 28 Myosins and Muscle Contraction Regulation until slide 11 Topic 21 Protein Synthesis and Targeting Transcription and Translation Eukaryotic Gene Structure Transcription A Gene is comprised of a protein coding region and a control region along the continuous DNA strand The control region contains regulatory DNA sequences o Regulatory transcription factors that are regulated by cell processes such as Lipophilic Hormone Receptors which bind to this regulatory region are repressors and keep the DNA sequence from being transcribed o Estrogen Response Element is an example of a promotor that binds to the control region and recruits RNA polymerase to transcribe DNA sequence into mRNA transcript gene or pre mRNA which includes the entire exon iron sequence for the gene which eventually becomes the mature mRNA that can code for protein in the ribosome The protein coding region between the exons are sequence regions called introns which do NOT code for protein These introns enable differential splicing of exons to encode for different protein isoforms contains sequence regions that are exons which code for protein and in Occurs in the nucleus where the DNA is located The copy of the gene that is the pre mRNA is made in the nucleus by the RNA polymerase complex which is further processed into mature mRNA There are several things that happen to the pre mRNA for processing into mature mRNA 1 Addition of a CAP to the 5 end 2 Addition of Poly A Tail to 3 end which adds a whole string of Adenine residues 3 Splicing removal of introns and splicing to attach the exon ends together a Splicing results in various different exon patterns which encode different versions of a protein different isoforms of that protein i Example Fibronectin is a protein located in the extracellular matrix The pre mRNA transcribed from the Fibronectin gene contains the entire intron and exon sequence which is then spliced differently depending upon the cell type in which this is occurring Above you can see that when the Fibronectin pre mRNA that contains exons and introns is processed into mature mRNA the introns black lines are spliced out and the exon ends are attached differently which can lead to some exons being spliced out and others remaining Different cells splice pre mRNA differently leading to multiple mature mRNA sequences In this case there is one which results from Fibroblasts which synthesize connective tissue and one that results from Hepatocytes liver cells ii Integrins are transmembrane receptors that are the bridges for cell to cell and cell to extracellular matrix interactions 1 Different integrins are in each type of cell The ingetrins bind to domains or specific exons In the case of Fibronectin the integrins bind to the EIIIA domain So if integrins only bind to Fibronectin s EIIIA domain then they will bind only to the fibroblast version of fibronectin mature mRNA and not the hepatocyte version of fibronectin mature mRNA 4 Once the above 3 steps occur the mature mRNA comes out of the nucleus through pore structures and into the cytoplasm for translation by Ribosomes Translation Mature mRNA is translated into the protein sequence by Ribosomes in the cytoplasm 1 Genetic Code 3 base code since this is the most conservative amount of bases that can code for more than 20 AA s a 1 base AA 41 4 possible AA s 20 which is necessary so has to be more b 2 bases AA 42 16 possible AA s 20 which is necessary so has to be more c 3 bases AA 43 64 possible AA s 20 which is necessary So this is the lowest possible amount of bases that will code for an entire protein which consists of 20 types of AA s d There are 3 bases nucleotides per codon and each codon codes for one AA Some AA s are encoded by more than one codon e AUG codes for Methionine and is the start codon while UGA UAA UAG are stop codons Ribosomes contain protein and some rRNA that do not contain protein coding sequences they only contribute to the structure of the ribosome and function as the enzyme that forms the peptide bond So RNA can function as a catalyst One region of the ribosome assembles on to the 5 end of the mRNA then scans down along the sequence until it finds the start codon AUG which codes for Methionine the first AA of the new peptide chain At this point the ribosome large and small subunits assemble together and begin synthesizing the new protein This is where mutations can come into play when an additional base is added or subtracted and the reading frame is shifted this will lead to the production of a different protein sequence or a premature stop codon can lead to a very short protein sequence As the ribosome reads each codon it matches the mRNA codon to the tRNA anticodon which carries the necessary AA via tRNA synthetase Ribosomes have 2 positions where the anticodon sequence of the tRNA can be matched to the codon on the mRNA There is a P site and A site o The P site anchors the elongating peptide to the ribosome o The A site is the open site where the next incoming tRNA with the new AA is brought in o Once the AUG codon is recognized in the P site and the tRNA with it s anticodon pairs with it the next tRNA anticodon AA comes in and binds at the A site Then the peptide bond is formed between the two AA and the bond is broken between the tRNA and Methionine and the Ribosome shifts what was in the A site to the P site allowing the next tRNA anticodon AA in then this repeats leaving you with a long chain of AA s o Once one of the three stop codons is recognized the ribosome dissociates from mRNA and releases the newly synthesized peptide Topic 22 Protein Synthesis and Targeting ER The newly synthesized protein made in the cytoplasm folds up into secondary tertiary or quaternary structures stabilized by H bonds Certain proteins remain in the cytoplasm while others are targeted to various other destinations Some are inserted into the membrane of the ER or they travel across the membrane of the ER and through the Golgi apparatus and some