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Chapter 7. Proteins: the buck stops here Prepared by Woojoo Choi Introduction to proteins 1) Protein – biological polymers that carry out most of the cell’s functions. – made from a linear chain of monomers, known as amino acids – folded into a variety of complex 3-D shapes – a chain of amino acids is called a polypeptide chain 2) What’s the difference between a polypeptide chain and a protein? – some protein consists of more than one polypeptide chain Introduction to proteins Role of proteins in the cell 1) Structural proteins – A protein that forms part of a cellular structure Role of proteins in the cell 2) Enzymes – A protein that carries out a chemical reaction – First binds its substrate, and then performs some chemical operations with it – Active site: special site or pocket on a protein where the chemical reaction occurs Role of proteins in the cell 2) Enzymes Role of proteins in the cell – Analog: molecules resembling natural substances well enough to fool the enzyme that use them – Some analogs inhibit the enzyme • Competitive inhibitor: chemical substance which inhibits the action of an enzyme by mimicking the true substrate well enough to be mistaken for it. – Other analogs do react • ONPG: an artificial substrate which releases a yellow color when split by the enzyme β-galactosidase • X-gal: split by β-galactosidase into a blue dye and galactose(Ch16) Role of proteins in the cell 3) Regulatory proteins – A protein that controls the expression of a gene or the activity of another protein 4) Transport proteins – A protein that carries other molecules across membranes or around the body How are proteins Constructed? 1) Amino acid – The monomer or subunit from which proteins are built – There are 20 different amino acids. – They all have a central carbon atom, the alpha carbon, surrounded by the four features. – R group: chemical group forming side chain of amino acid Formation of polypeptide chains 1) Amino acids are joined together by peptide bonds to give a polypeptide chain – Amino- or N-terminus: the end of a polypeptide chain that is made first and has a free amino group – Carboxyl- or C-terminus: the end of a polypeptide chain that is made last and has a free carboxy group Three-dimensional structures 1) Primary structure – The linear order in which subunits of a polymer are arranged 2) Secondary structure – Initial folding of a polymer due to hydrogen bonding Three-dimensional structures – There are two alternative secondary structures alpha-helix and betasheet Three-dimensional structures 3) Tertiary structure – Final 3-D folding of a polymer chain – The polypeptide chain, with its performed regions of a-helix and bsheet, to give the final 3-D structure. – The level of folding depends on the side chains of 20 different amino acids – 3-D folding is the result of two factors, hydrophilic and hydrophobic. – Some side chains are hydrophilic and others are hydrophobic. Three-dimensional structures Three-dimensional structures Quaternary structure of proteins 4) Quaternary structure – Aggregation of more than one polymer chain in final structure – Not all proteins have more than one polypeptide chain – The hydrophilic and hydrophobic forces are responsible Twenty different amino acids 1) 20 different amino acids allow for a great variety of 3-D structure and of chemical reactivity Amino Acid 3-Letter Code 1-Letter Code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Gultamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Twenty different amino acids Amino Acid 3-Letter Code 1-Letter Code Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V How are proteins made? 1) Translation – Making a protein using the information provided by mRNA – Central dogma: basic plan of genetic information flow in living cells that relates genes(DNA), RNA, and proteins Decoding the genetic code 1) Codon – Group of three RNA and DNA bases which encodes a single amino acid – Some amino acids are encoded by more than one codon Ribosome – The cell’s decoding machine 1) Ribosome – The cell’s machinery for making proteins – Bind mRNA and translate it, so synthesizing a polypeptide chain. – Consist of two subunits, 50s and 30s How may tRNA 1) To – – – read the codons, transfer RNA(tRNA) is needed tRNA: RNA molecule that carry amino acids to ribosome tRNA has an anticodon Anticodon: group of three complementary bases that recognize and bind to a codon on the mRNA How does the tRNA get its amino acid? 1) Amino-acyl tRNA synthetase: enzyme that attaches an amino acid to tRNA 2) Uncharged tRNA: tRNA without an amino acid attached 3) Charged tRNA: tRNA with an amino acid attached Structure of transfer RNA 1) 2) 3) 4) 5) 6) Typical tRNA: 4 short base-paired stems, 3 loops At acceptor stem: amino acid In the anticodon loop: anticodon TψC-loop: contains pseudouracil D-loop: contains dihydrouracil In real life, the tRNA cloverleaf is folded up further Reading Frames 1) Start codon – the special AUG codon that signals that start of a protein Reading Frames GAAAUGUAUGCAUGCCAAAGGAGGCAUCUAAGGA 111 222 333 444 555 666 777 888 111 222 333 444 555 666 777 888 111 222 333 444 555 666 777 888 2) Reading frame – One of three possible ways to read off the bases of mRNA in groups of three so as to give codons 3) Open reading frame – Sequence of mRNA that can be translated to give a protein – Any mRNA will have several possble ORFs and we have to find correct one Getting protein synthesis started 1) fMet or formyl methionine – Chemically tagged version of methionine used to start the polypeptide chain in prokaryotic cells 2) Ribosome binding site – Sequence on mRNA at the front of the message which is recognized by the ribosome (Shine Delgarno sequence) 3) Anti-Shine-Dalgarno sequence – Sequence on 16S rRNA that is complementary to the Shine Delgarno sequence Getting protein synthesis started – A (acceptor) site: binding site on the ribosome for the tRNA which brings in the next amino acid – P (peptide) site: binding site on the ribosome for the tRNA that is holding the growing polypeptide chain Elongation of a growing protein The elongation factors 1) Elongation factors: Proteins that oversee the elongation of a growing polypeptide chain 2) EF-T (EF-Tu and EF-Ts): The tRNA is delivered to the ribosome and instaled into the A-site 3) EF-G: moving everybody sideways Termination of protein synthesis 1) Stop codon – Codon that signals the end of a protein – UGA, UAG, and UAA 2) Releas factors – Proteins that supervise the release of a finished polypeptide chain from the ribosome One mRNA can code for several proteins 1) In bacteria several protein may be encoded by the same messenger RNA 2) Cistron – Segment of DNA that encodes a single polypeptide chain 3) Polycistronic mRNA – mRNA carrying multiple cistron and which may be translated to give several different protein molecule. Several ribosomes can read the same message at once 1) Polysome (polyribosome) – Group of ribosomes that bind to and translate the same mRNA Coupled translation and transcription in bacteria 1) Coupled transcription-translation – When ribosomes of bacteria start translating an mRNA which is still being transcribed from the genes (In eukaryotic cells, this is verboten, because the DNA is inside the nucleus and the ribosomes are outside.) Some proteins come to a bad end 1) When ribosomes receive a defective mRNA, they have problems 2) If there is no stop codon, a ribosome will just sit there forever and trapped Some proteins come to a bad end tmRNA – a special RNA used to terminate protein synthesis when it find a ribosome stalled by a bad mRNA – It acts partly like tRNA and mRNA – Leave the short strech of amino acids added to the end of the defective protein – The short stretch act as a signal which is recognized by tail specific protease. – Tail specific protease: enzyme that destroys mismade proteins by eating them tail first