Molecular Biology PROTEIN SYNTHESIS

Proteins DNA codes for proteins Proteins provide phenotypic traits Genotype – genetic make up Phenotype – physical characteristics or specific traits Genes are found on chromosomes The genes provide the instructions for making proteins DNA  RNA  Protein Synthesis

There are 2 steps to protein synthesis Transcription – the transfer of genetic information from DNA into a RNA molecule Translation – transfer of RNA into a protein

DNA Nucleus Cytoplasm Figure 10.6A Flow of genetic information in a eukaryotic cell. Transcription is the production of RNA using DNA as a template. In eukaryotic cells, transcription occurs in the nucleus, and the resulting RNA (mRNA) enters the cytoplasm. Translation is the production of protein, using the sequence of nucleotides in RNA. Translation occurs in the cytoplasm for both prokaryotic and eukaryotic cells.

DNA Transcription RNA Nucleus Cytoplasm DNA Transcription RNA Nucleus Cytoplasm Figure 10.6A Flow of genetic information in a eukaryotic cell. Transcription is the production of RNA using DNA as a template. In eukaryotic cells, transcription occurs in the nucleus, and the resulting RNA (mRNA) enters the cytoplasm. Translation is the production of protein, using the sequence of nucleotides in RNA. Translation occurs in the cytoplasm for both prokaryotic and eukaryotic cells.

DNA Transcription RNA Nucleus Cytoplasm Translation Protein DNA Transcription RNA Nucleus Cytoplasm Figure 10.6A Flow of genetic information in a eukaryotic cell. Transcription is the production of RNA using DNA as a template. In eukaryotic cells, transcription occurs in the nucleus, and the resulting RNA (mRNA) enters the cytoplasm. Translation is the production of protein, using the sequence of nucleotides in RNA. Translation occurs in the cytoplasm for both prokaryotic and eukaryotic cells. Translation Protein

Archibold Garrod 1909 Suggested genes dictate phenotypes through enzymes He studied inherited diseases He said diseases reflected a person’s inability to make an enzyme Ex – alkaptonuria causes urine to be black because of the absence of an enzyme to break down alkapton

Beadle and Tatum 1940s They worked with bread mold – Neurospora crassa They studied strains of mold that could not grow on a simple growth medium Each of these nutritional mutants lacked an enzyme in a metabolic pathway that make some molecule the mold needed (amino acid) Showed each mutant was defective in a single gene This led to the 1 gene – 1 enzyme hypothesis

One gene – One polypeptide Hypothesis One gene –one enzyme became one gene – one protein hypothesis This is because not all proteins are enzymes This hypothesis soon became one gene – one polypeptide This is because many proteins are made up of two or more polypeptide chains

Codons The sequence of nucleotides in DNA provides a code for constructing a protein Protein construction requires a conversion of a nucleotide sequence to an amino acid sequence The information comes from genes Genes - section of DNA that is made up of hundreds or thousands of nucleotides in a specific order

Codons 1 codon is made up of 3 nucleotides Each amino acid is specified by a codon 1 codon is made up of 3 nucleotides 64 codons are possible Some amino acids have more than one possible codon CAGAUUUAUCCG – How many codons are here?

Transcription and Translation Transcription – a gene is copied and a complementary RNA strand is formed Remember there is no thymine in RNA instead there is URACIL Rewrites the DNA code into RNA, using the same nucleotide “language” Translation – converts the RNA strand into a protein Remember that amino acids are the monomers of proteins Switching from the nucleotide “language” to amino acid “language”

DNA molecule Gene 1 Gene 2 Gene 3 DNA strand Transcription RNA Codon DNA molecule Gene 1 Gene 2 Gene 3 DNA strand Figure 10.7 Transcription and translation of codons. Transcription RNA Codon Translation Polypeptide Amino acid

DNA strand Transcription RNA Codon Translation Polypeptide Amino acid DNA strand Transcription RNA Codon Translation Figure 10.7 Transcription and translation of codons. Polypeptide Amino acid

Genetic Codes Genetic code – the set of rules giving the information between codons in RNA and amino acids of proteins Three nucleotides specify one amino acid 61 codons correspond to amino acids AUG codes for methionine and signals the start of transcription 3 “stop” codons signal the end of translation (UAA, UAG, UGA) Redundant: More than one codon for some amino acids

Genetic Codes “Wobble position” – the first two bases count more than the third amino acid in a codon. Nearly universal so evolved early on in life

Second base First base Third base First base Third base Figure 10.8A Dictionary of the genetic code (RNA codons). This listing of the codon “dictionary” can be used to illustrate the triplet and redundant nature of the code. While methionine and tryptophan have only one codon each, leucine, serine, and arginine each have six codons. It can also be pointed out that codons for the same amino acid often differ in the third nucleotide, a phenomenon described as “wobble.” The base pairing of the first two nucleotides of the codon with corresponding positions in the anticodon is stringent, but pairing of the third is weaker and more flexible. The wobble hypothesis proposed by Francis Crick allows for some nonstandard pairings that account for some of the redundancy of the genetic code. For example, if the third position of the codon is a U or C, it can pair with a G on the anticodon. This would mean that one tRNA, rather than two, could be used to translate UUU and UUC, for example. Estimates of 30–50 tRNAs necessary to pair with 61 codons are borne out by studies that identify 45 different tRNAs in some cell types.

Strand to be transcribed DNA Figure 10.8B Deciphering the genetic information in DNA.

Strand to be transcribed DNA Transcription RNA Figure 10.8B Deciphering the genetic information in DNA. Start codon Stop codon

Second base First base Third base First base Third base Figure 10.8A Dictionary of the genetic code (RNA codons). This listing of the codon “dictionary” can be used to illustrate the triplet and redundant nature of the code. While methionine and tryptophan have only one codon each, leucine, serine, and arginine each have six codons. It can also be pointed out that codons for the same amino acid often differ in the third nucleotide, a phenomenon described as “wobble.” The base pairing of the first two nucleotides of the codon with corresponding positions in the anticodon is stringent, but pairing of the third is weaker and more flexible. The wobble hypothesis proposed by Francis Crick allows for some nonstandard pairings that account for some of the redundancy of the genetic code. For example, if the third position of the codon is a U or C, it can pair with a G on the anticodon. This would mean that one tRNA, rather than two, could be used to translate UUU and UUC, for example. Estimates of 30–50 tRNAs necessary to pair with 61 codons are borne out by studies that identify 45 different tRNAs in some cell types.

Strand to be transcribed DNA Transcription RNA Figure 10.8B Deciphering the genetic information in DNA. Start codon Stop codon Translation Polypeptide Met Lys Phe

Transcription Happens in the nucleus of eukaryotic cells An RNA molecule is made from DNA because DNA cannot leave the nucleus The DNA strands separate but only one acts as a template Complementary RNA nucleotides to the DNA template strand are added to form the mRNA strand

Transcription RNA polymerase – enzyme that helps add RNA nucleotides Remember there is no thymine in RNA The Promoter A DNA sequence has a “start transcribing” signal made up of a specific nucleotide sequence RNA polymerase binds to the promoter The promoter also tells which DNA strand is the template

Steps of Transcription Initiation Attachment of RNA polymerase to promoter Start of RNA synthesis Elongation RNA nucleotides continue to be attached The two strands which are being held together by H bonds start to separate and the DNA molecules retwist

Steps of Transcription Termination The RNA polymerase reaches the terminator Terminator – a sequence that signals the end of the gene RNA polymerase detaches.

RNA nucleotides RNA polymerase Direction of transcription Template RNA nucleotides RNA polymerase Figure 10.9A A close-up view of transcription. Direction of transcription Template strand of DNA Newly made RNA

Transcription Animation RNA polymerase DNA of gene Promoter Terminator DNA of gene Promoter DNA Terminator DNA 1 Initiation Area shown in Figure 10.9A 2 Elongation Figure 10.9B Transcription of a gene. Growing RNA 3 Termination Transcription Animation Completed RNA RNA polymerase

Modification of mRNA Messenger RNA (mRNA) contains codons for protein sequences The mRNA strand is processed before it leaves the nucleus through a nuclear pore A cap (5’ end: single guanine nucleotide) and a tail added to (3’ end: Poly-A tail of 50–250 adenines) is added to the mRNA strand This helps with the export of mRNA Protects the mRNA from being degraded by enzymes Helps the mRNA strand to bind to the ribosomes

Modification of mRNA Introns have to be removed Genes do have some noncoding areas These noncoding sequences are called introns Exons – are the coding regions of the gene These are the parts of the gene that are expressed as amino acids Introns get removed and exons get joined together This cutting and pasting is called RNA splicing

Addition of cap and tail Cap Exon Intron Exon Intron Exon DNA Transcription Addition of cap and tail Cap RNA transcript with cap and tail Introns removed Tail Exons spliced together mRNA Coding sequence Figure 10.10 The production of eukaryotic mRNA. Nucleus Cytoplasm

Transfer RNA (tRNA) Helps to read mRNA to make a protein Amino acids are found in the cytoplasm tRNAs help to transfer these amino acids to help make proteins tRNA has to match the amino acid to the codon on the mRNA strand

Transfer RNA (tRNA) Structure Made up of a single strand of RNA made up of about 80 nucleotides Folds and twists on itself At one end is the anticodon – special triplet of bases that is complementary to a codon on the mRNA strand The other end has the site for an amino acid to attach There is an enzyme here that makes sure to join the correct amino acid to the tRNA molecule

Amino acid attachment site Hydrogen bond RNA polynucleotide chain Figure 10.11A The structure of tRNA. tRNA takes on its characteristic shape as a result of hydrogen bonding between bases on the same RNA chain. Anticodon

Figure 10.11B A molecule of tRNA binding to an enzyme molecule (blue). The specificity of the genetic code depends on enzymes called tRNA synthetases that recognize the anticodon and catalyze the reaction that attaches the appropriate amino acid to the opposite end of the tRNA. ATP provides energy for the reaction.

Ribosomes Helps with the making of the protein They are found in the cytoplasm Made up of 2 subunits Made up of ribosomal RNA (rRNA) They have a binding site for mRNA and two binding sites or tRNA

Growing polypeptide tRNA molecules Large subunit mRNA Small subunit Growing polypeptide tRNA molecules Large subunit Figure 10.12A The true shape of a functioning ribosome. This figure emphasizes the positioning of the small and large ribosomal subunits, along with mRNA and tRNA molecules. mRNA Small subunit

tRNA-binding sites Large subunit mRNA binding site Small subunit tRNA-binding sites Large subunit mRNA binding site Figure 10.12B Binding sites of a ribosome. This figure locates the binding sites for mRNA and tRNAs on the ribosome. Small subunit

Next amino acid to be added to polypeptide Growing polypeptide tRNA Next amino acid to be added to polypeptide Growing polypeptide tRNA mRNA Figure 10.12C A ribosome with occupied binding sites. This figure shows that one of the tRNA binding sites (P site) holds the growing peptide chain while the adjacent site (A site) holds the tRNA carrying the next amino acid to be added to the chain. Codons

Steps to Translation 1. Initiation mRNA, first the tRNA molecule with Met, and both subunits of the ribosome come together The start codon reads AUG and codes for methionine The first tRNA has the anticodon UAC The two ends of the mRNA strand have bases that help attach it to the ribosomes mRNA binds to the small ribosomal subunit tRNA binds to the start codon

Start of genetic message Start of genetic message Figure 10.13A A molecule of mRNA. This figure shows that the bases of the codons are arranged linearly along an mRNA. End

Steps to Translation The large ribosomal subunit binds to the small subunit The first tRNA is in one of the two tRNA binding sites on the ribosomes This is the P site. P site – holds the growing polypeptide chain A site – is vacant and is waiting for the next tRNA molecule

Met Met Large ribosomal subunit Initiator tRNA P site A site Start Met Met Large ribosomal subunit Initiator tRNA P site A site Start codon mRNA Figure 10.13B The initiation of translation. The two-step process of initiation is shown in this figure. In prokaryotic cells, the binding of the first tRNA, formyl-methionine (f-met) tRNA has been shown to stabilize the initiation complex. Small ribosomal subunit 1 2

Steps to Translation 2. Elongation Amino acids are added one by one to the first amino acid (Met) a 3 step process: 1. Codon recognition: next tRNA’s anticodon binds to the complementary codon on the mRNA at the A site 2. Peptide bond formation: joining of the new amino acid to the chain Amino acids on the tRNA at the P site are attached by a covalent bond to the amino acid on the tRNA at the A site The ribosome helps with the bonding

Steps to Translation Elongation (Con’t) 3. Translocation - tRNA is released from the P site and the ribosome moves tRNA from the A site into the P site So the next codon on the mRNA is in the A site.

Steps to Translation 3. Termination Elongation continues until a stop codon (UAA, UAG, UGA) A release factor adds on a water instead of an amino acid Everything breaks apart

Amino acid Polypeptide P site A site Anticodon mRNA Codons 1 Codon recognition Figure 10.14 Polypeptide elongation.

Amino acid Polypeptide P site A site Anticodon mRNA Codons 1 Codon recognition Figure 10.14 Polypeptide elongation. 2 Peptide bond formation

Translation Animation Amino acid Polypeptide P site A site Anticodon 1 Codon recognition Codons Amino acid Anticodon P site mRNA 2 Peptide bond formation 3 Translocation New peptide bond Figure 10.14 Polypeptide elongation. Translation Animation

Amino acid Polypeptide P site A site Anticodon mRNA Codons 1 Codon recognition mRNA movement Stop codon Figure 10.14 Polypeptide elongation. 2 Peptide bond formation New peptide bond 3 Translocation

Review Does translation represent: DNA  RNA or RNA  protein? Where does the information for producing a protein originate: DNA or RNA? Which one has a linear sequence of codons: rRNA, mRNA, or tRNA? Which one directly influences the phenotype: DNA, RNA, or protein? Does translation represent: DNA  RNA or RNA  protein? Answer: RNA  protein Where does the information for producing a protein originate: DNA or RNA? Answer: DNA Which one has a linear sequence of codons: rRNA, mRNA, or tRNA? Answer: mRNA Which one directly influences the phenotype: DNA, RNA, or protein? Answer: protein

Figure 10.15 Summary of transcription and translation. DNA mRNA is transcribed from a DNA template. 1 mRNA RNA polymerase Amino acid Translation Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. 2 Enzyme ATP tRNA Figure 10.15 Summary of transcription and translation. This figure is an overview of the processes, emphasizing the participation of enzymes and the requirement of an energy source. Beyond its use for attaching amino acids to tRNAs, phosphate-bond energy is also needed during initiation, elongation, and termination of translation. The need to obtain amino acids from the diet could be described along with this slide. If amino acids are present in sufficient amounts, they will be continually attached to tRNAs and available for protein synthesis. If one or more amino acids is low in quantity, the translation of any protein containing those amino acids will be terminated prematurely when the corresponding codon is reached and a tRNA fails to bind to the A site. For humans, animal protein sources have an appropriate profile of essential amino acids. Plant proteins can be combined to provide an adequate amino acid balance. Anticodon Initiator tRNA Large ribosomal subunit Initiation of polypeptide synthesis 3 The mRNA, the first tRNA, and the ribosomal sub-units come together. Start Codon Small ribosomal subunit mRNA

to the polypeptide chain as the mRNA is moved through the ribosome, New peptide bond forming Growing polypeptide 4 Elongation A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time. Codons mRNA Polypeptide Figure 10.15 Summary of transcription and translation. This figure is an overview of the processes, emphasizing the participation of enzymes and the requirement of an energy source. Beyond its use for attaching amino acids to tRNAs, phosphate-bond energy is also needed during initiation, elongation, and termination of translation. The need to obtain amino acids from the diet could be described along with this slide. If amino acids are present in sufficient amounts, they will be continually attached to tRNAs and available for protein synthesis. If one or more amino acids is low in quantity, the translation of any protein containing those amino acids will be terminated prematurely when the corresponding codon is reached and a tRNA fails to bind to the A site. For humans, animal protein sources have an appropriate profile of essential amino acids. Plant proteins can be combined to provide an adequate amino acid balance. 5 Termination The ribosome recognizes a stop codon. The polypeptide is terminated and released. Stop codon

Mutations Any change in the nucleotide sequence of DNA Divided into 2 general categories 1. Point Mutations (Base Substitutions - replacement on one nucleotide with another (CAG  CAA) Silent – A point mutation that doesn’t not cause a change in the function of the final protein Missense – A point mutation that changes the codon to code for a different amino acid, resulting in a nonfunctioning protein Nonsense – A point mutation that changes the sequence to code for a premature stop codon.

Mutations 2. Frameshift Mutations – “Reading Frame” of the sequence has been altered. Insertion Mutation – One or more nucleotides are inserted into the sequence. CAAGAC  CAATGAC Deletion Mutation – One or more nucleotides have been removed from the sequence CAAGAC  CAAGC

Mutations Duplication Mutation – A region of a chromosome is copied, resulting in multiple copies of that region Inversion Mutation –The order of a segment of chromosome is reversed. Mutagenesis – production of mutations Can be spontaneous during DNA replication Can also result from mutagens – physical or chemical agent that can cause mutations (ex- radiation) Carcinogen – A mutagen that causes cancer

Normal gene mRNA Protein Met Lys Phe Gly Ala Base substitution Met Lys mRNA Protein Met Lys Phe Gly Ala Base substitution Met Lys Phe Ser Ala Figure 10.16B Types of mutations and their effects. This figure contrasts the multiple amino acid changes caused by a deletion with the single amino acid change caused by a substitution. Base deletion Missing Met Lys Leu Ala His