Biotechnology and Protein Production in Bacteria and Fungi PDF

By Noura EI-Ahmady El-Naggar Professor of Biotechnology, Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, New Borg El-Arab City  Topics of the lecture...

By Noura EI-Ahmady El-Naggar Professor of Biotechnology, Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, New Borg El-Arab City  Topics of the lecture  Introduction to proteins  Protein structure  From Gene to Protein Remember Proteins? They are one of the vital biomolecules of life. Proteins perform a variety of essential processes to sustain an organism's survival. The building blocks of proteins are called amino acids, they are made up of a chain of amino acid monomers. Amino acids in a protein chain are linked by peptide bonds.  Amino Acid Structure  An amino acid is a group of organic molecules that consists of a basic amino group (-NH2), an acidic carboxyl group (-COOH) and between them a carbon atom.  Each molecule contains a central carbon (C) atom, called the α- carbon, to which both an amino and a carboxyl group are attached.  The side chain, known as "the R group," gives the amino acid its identity and contributes to the structure and function of the  protein. There are 20 amino acids that make up proteins and all have the same basic structure, differing only in the R-group or side chain they have. The simplest, and smallest, amino acid is glycine for which the R-group is a hydrogen (H). Table 1: Shows amino acid abbreviations, 3-letters and single letter codes used for the 20 amino acids found in proteins. Amino Acid 3-Letter 1-Letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Amino Acid 3-Letter 1-Letter Cysteine Cys C Leucine Leu L Glutamic acid Glu E Lysine Lys K Glutamine Gln Q Methionine Met M Glycine Gly G Phenylalanine Phe F Histidine His H Isoleucine Ile I Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V  Structural organization of protein Proteins are structurally organized into four levels; primary structure, secondary structure, tertiary structure and quaternary structure. 1.1 Primary structure of protein: Primary structure of protein refers to linear sequence of amino acids in a polypeptide chain or protein. -COOH group (carboxyl group) of one amino acid is linked with -NH2 group of other amino acid by peptide bond. The primary structure of a protein is reported starting from the end at which – NH2 group is free (called amino-terminal (N) end) to the C-terminal (C) end (the end at which –COOH group is free).  Level of structural organization of protein 2 Secondary structure of protein: 2.  Amino acids that are located near to each other interacts to form regular arrangement called secondary structure.  Formation of secondary structure involve local folding of polypeptide chain.  There are three commonly occurring secondary structure. They are α-Helix, β-sheet and β-bends or β-turn. α-helix structure:  α-helix is a sequence of amino acids in a protein that are twisted into a right handed spiral structure.  In α-helix, -C=O group of each amino acid is hydrogen bonded with –NH group of other amino acid which is situated four amino acid ahead. Therefore, -C=O and –NH group of all amino acids are hydrogen bonded in α-helical structure.  R-group of amino acid in α-helix are projected outside of the spiral to minimize steric hindrance. β-sheet structure: β-Sheet is the most stable form of secondary structure of protein. It is formed between two different polypeptide chains which are placed parallel or antiparallel to each other. β-sheet structure: In a parallel β-sheet, the polypeptide chains run in the same direction. This means that the N-terminus of one strand aligns with the N-terminus of the neighboring strand, and the C-terminus of one strand aligns with the C-terminus of the neighboring strand. whereas in an antiparallel β-sheet, the polypeptide chains run in opposite directions. This means that the N-terminus of one strand aligns with the C-terminus of the neighboring strand, and vice versa. In β-sheet structure, two polypeptide backbones are linked with each other by H- bond which are formed between –CO and –NH group. R-group of amino acids are alternately projected above and below the β-sheet. The surface of β-sheet is not straight but it is pleated. Therefore, it is also known as β-pleated sheet. It can also be formed by folding of same polypeptide chain. β-bends or β-turn structure: A β-turn is composed of four amino acids. In a β-turn, a tight loop is formed when the C=O group of the first amino acid forms a hydrogen bond with the N–H group of the fourth amino acid. Glycine and proline are always found in β-bend structure. β-turn structure 3.3 Tertiary structure of protein:  The tertiary structure of a protein is the three dimensional shape that comes from interactions between different secondary structures.  The tertiary structure have a single polypeptide chain "backbone" with one or more protein secondary structures (α-helix, or β-sheet or β-bend).  Amino acid side chains and the backbone interact and Tertiary structure bond in a number of ways like H-bond, hydrophobic of protein interaction, ionic bond and disulphide bond. 4 Quaternary structure of protein:  The quaternary structure is the three-dimensional structure consisting of the aggregation of two or more individual proteins (usually called protein subunits into a closely packed arrangement called the quaternary structure of proteins.  The separate protein chains aggregate with self to form homodimers, homotrimers, or homopolymers or aggregate with different proteins to form heteropolymers.  4 Quaternary structure of protein:  Bonds like H-bond, ionic bond, hydrophobic interaction helps to form quaternary structure.  The hemoglobin proteins possess heterogeneous quaternary structure because it is made up of 2 α chains and two β-chains.  In collagen, the basic structural unit is a triple-stranded helical molecule. Triple-stranded polypeptide molecules pack together side by side to give a crystalline fiber. Quaternary structure of protein  “From Gene to Protein” The instructions for making proteins are found in DNA. How are cell proteins made from the instructions in DNA ? Proteins Cells DNA  The bacterial chromosome is usually a single, circular double-stranded DNA molecule. There are extra circles called plasmids.  A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently.  The function of the bacterial chromosome is to carry the genetic information for bacteria  DNA is organized into a superhelical structure called the nucleoid (meaning nucleus-like), which is not enclosed within a membrane-bound nucleus like in eukaryotes.  To fit within the bacterial cell, the bacterial nucleoid (a bacterial genome) must be compacted about a 1000-fold by nucleoid binding proteins (DNA- binding proteins). This involves the formation of loop domains. The number of loops varies according to the size of the bacterial chromosome and the species.  A typical bacterial chromosome contains a few thousand different genes. Prokaryotic chromosomes  Eukaryotic cells including Fungi contain large amount of genomic DNA tightly packaged in chromosomes contained within a specialized organelle, the nucleus. Chromosomes contain both DNA and protein.  The proteins that bind to the DNA to form eucaryotic chromosomes are traditionally divided into two general classes: the histones and the nonhistone chromosomal proteins. The complex of both classes of protein with the nuclear DNA of eucaryotic cells is known as chromatin.  In addition to nuclear DNA, some DNA is present in mitochondria.  DNA, is a double stranded nucleic acid carries the genetic instructions.  Proteins are synthesized by the ribosomes using messenger RNA transcribed from DNA.  RNA is a single stranded molecule and it is transcribed (synthesized) from DNA by enzymes called RNA polymerases.  Transcription is a way of taking the information from DNA, and making RNA. Eukaryotic chromosomes  Protein Synthesis A comparison of the helix and base structure of RNA and DNA Comparison DNA RNA Full Name Deoxyribonucleic Acid Ribonucleic Acid Function DNA replicates and stores genetic information RNA converts the genetic information contained within an organism contained within DNA to a format used to build proteins. Types DNA mainly are of two major types i.e., nuclear The three types of RNA are M DNA and plasmid DNA which are also known as (messenger)-RNA, R(ribosomes)- chromosomal and extrachromosomal DNA RNA, T(transfer)-RNA Structure of DNA consists of two strands, arranged in a RNA only has one strand, but like molecules double helix. These strands are made up of DNA, is made up of nucleotides. RNA subunits called nucleotides. Each nucleotide strands are shorter than DNA strands. contains a phosphate, a 5-carbon sugar molecule and a nitrogenous base. Sugar The sugar in DNA is deoxyribose, which contains RNA contains ribose sugar molecules, one less hydroxyl group than RNA’s ribose. with a hydroxyl group. Bases The bases in DNA are Adenine (‘A’), Thymine RNA shares Adenine (‘A’), Guanine (‘T’), Guanine (‘G’) and Cytosine (‘C’). (‘G’) and Cytosine (‘C’) with DNA, but contains Uracil (‘U’) rather than Thymine. Base Pairs Adenine and Thymine pair (A-T). Cytosine and Adenine and Uracil pair (A-U). Guanine pair (C-G) Cytosine and Guanine pair (C-G) Comparison DNA RNA Replication DNA possesses the nature of self-replication RNA does not tend to replicate and when as it replicates on its own without any prior required, RNA replication occurs in the support. cytoplasm. Length DNA is a much longer than RNA. A RNA molecules are variable in length, but chromosome is a single, long DNA much shorter than long DNA polymers. A molecule, which would be several large RNA molecule might only be a few centimetres in length when unravelled. thousand base pairs long. Stability Due to its deoxyribose sugar, which contains RNA, containing a ribose sugar, is more one less oxygen-containing hydroxyl group, reactive than DNA and is not stable in DNA is a more stable molecule than RNA, alkaline conditions. RNA is more easily which is useful for a molecule which has the subject to attack by enzymes. task of keeping genetic information safe. Location DNA is found in the nucleus, with a small RNA forms found in the nucleus or amount of DNA also present in ribosomes and cytoplasm depending on the mitochondria. type of RNA formed. Ultraviolet DNA can be damaged due to the radiation RNA is more resistant to damage from UV Sensitivity produced by the ultraviolet rays. light than DNA.  Three main types of RNA 1. Messenger RNA (abbreviated mRNA) is a type of single-stranded RNA involved in protein synthesis. mRNA is made from a DNA template during the process of transcription. The role of mRNA is to carry protein information from the DNA in a cell’s nucleus to the sites of protein synthesis in the cytoplasm (the ribosomes). 2. Ribosomal RNA (abbreviated rRNA) is the major component of ribosomes, which are responsible for protein synthesis. 3. Transfer RNA (abbreviated tRNA) - Transfers amino acids to ribosomes during protein synthesis. Transcription  Transcription is the process through which a DNA sequence is transcribed to produce a mRNA.  During transcription, the DNA splits into two strands. Only one strand of a DNA molecule is transcribed to mRNA; this strand is called antisense strand or template strand and the RNA produced is termed as sense RNA.  The other strand of the DNA duplex is known as coding strand or sense strand (not used to synthesize RNA).  Transcription proceeds from the 3’ end to the 5’ direction end of the template.  Transcription  This done by first opening the double helix with an enzyme called DNA Helicase.  Another enzyme called RNA polymerase will match new bases to the original DNA attaching them in a long strand of mRNA.  When the enzyme reaches the end, the strand will be removed and the DNA can close.  Once the DNA has been transcribed to produce mRNA, the mRNA can find a Ribosome.  The mRNA arranged in codons, each code for an amino acid which are the building blocks of proteins.  Genetic code  Genetic code, the sequence of nucleotides in DNA and RNA that determines the amino acid sequence of proteins.  Protein sequences consist of 20 commonly occurring amino acids; therefore, it can be said that the protein alphabet consists of 20 letters. There are only four nucleotides involved in the structure of both DNA and RNA, there must be at least 20 different genetic codes to specify the 20 amino acids. The number of nucleotides that are responsible for the formation of the amino acid code: If we consider that the genetic code is: 1. Single (One letter): each nucleotide represents an amino acid code, so the possible code words are 4 codes, then they form 4 amino acids only (A, C, G and U) (this is impossible). 2. Double (Two letters): each two nucleotides represent an amino acid code, in all possible combination of any two nucleotides, it gives 4² = 16 different code words (this is impossible). 3. Triple (three letters): Using a three-nucleotide code means that the number of codes will be 4³ = 64 different code words, then there is more than one codon for most of the amino acids, except methionine.  The genetic code for translating each nucleotide triplet, or codon, in mRNA into an amino acid or a termination signal in a nascent protein.  There is only one codon for the starting of the protein synthesis which is called “start codon” that encodes methionine and marks a protein’s beginning (AUG).  There are three “stop codons” (UAG, UGA and UAA) at which protein synthesis mechanism stops and gives a signal.  Protein synthesis – Translation The key components required for translation are mRNA, ribosomes, transfer RNA (tRNA) and various enzymatic factors. Ribosomes are responsible for synthesizing the proteins in all cells by a process called translation. It is called translation because ribosomes use the genetic information encoded in the mRNA and must "translate" and converts the message contained in the nucleotides of mRNAs into the polypeptide sequence Structure:  The general structure of ribosomes is the same in all cells, but ribosomes of prokaryotes as bacteria are smaller than ribosomes in the cytoplasm of eukaryotes as fungi.  The ribosome is a factory for protein synthesis in the cells and made of ribosomal RNA molecules and proteins (ribonucleoprotein complex).  A ribosome is composed of two subunits: large and small.  In prokaryotic, large subunits have a sedimentation rate of 50’s. Small subunits have a sedimentation rate of 30’s. To function, a small and large subunit must come together and its ribosomes sediment at 70’s. While larger eukaryotic ribosomes sediment at 80’s (The large 60’s subunit and whereas the small 40’s subunit.  The tRNA binding sites on a ribosome A-site (aminoacyl-tRNA site) An aminoacyl-tRNA (referred to as a charged tRNA) is the first binding site in the ribosome has an anticodon sequence complementary to the mRNA which binds the tRNA with the new amino acid to be added. P-site (peptidyl-tRNA site) is the second binding site for tRNA in the ribosome. During protein translation, the P-site holds the tRNA which is linked to the growing polypeptide chain. E-site (exit site, uncharged tRNA ) is the third and final binding site for t- RNA in the ribosome during translation. The E site is a location on the ribosome where the empty tRNA are released from the ribosome.  tRNA’s: free-roaming molecule that circulates in the cytoplasm. If it receives the coded instructions from the mRNA, it will then find and carry the amino acid that matches the mRNA codon in the cytoplasm to the ribosome to be built into proteins.  Translation of an mRNA molecule by the ribosome occurs in three stages: Initiation, Elongation and Termination Initiation of Translation  The initiator tRNA molecule carrying the amino acid methionine binds to the AUG start codon of the mRNA transcript at the ribosome’s P site where it will become the first amino acid incorporated into the growing polypeptide chain  A group of proteins called initiation factors (IFs) enable the small subunit of a ribosome to bind to an mRNA and form the initiation complex.  Protein synthesis is initiated with the initiation codon (START codon) AUG (the first codon in the transcribed mRNA that undergoes translation). AUG codes for the amino acid methionine (Met) in eukaryotes and formyl methionine (fMet) in prokaryotes and starts translation of mRNA.  After, the methionine-tRNA will bind to the AUG start codon, a ribosome large subunit joins this complex at the P site on the ribosome. This ends the initiation stage.  Elongation  During the elongation process, the tRNA carries an amino acid to the ribosomes. Following initiation, the first tRNA (for methionine) is located within the P site.  The second tRNA arrives and attaches at the A site, with the correct anticodon and the correct amino acid.  By an enzymatic reaction, the amino acids between the P and A chains are joined together by a peptide bond. Then, both tRNAs move one site forward (A to P; P to E). “Uncharged” or empty tRNA will prepare to exit from the E site (exit site).  The ribosome moves along the strand from the 5' end to the 3' end until is find a STOP codon, near the end of the mRNA, is reached. Termination  At the stop codon, a stop protein (release factor) is a specific sequence of nucleotides in mRNA that signals the termination of protein translation.  Termination happens when a stop codon in the mRNA (UAA, UAG, or UGA) enters the A site in place of tRNA.  This will cause the release of the last tRNA, the polypeptide chain and cause the ribosome to fall apart. AP Biology