Gene Lecture 12 Translation
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Fundamental Genetics Lecture 12
Translation and Proteins John Donnie A. Ramos, Ph.D. Department of Biological Sciences College of Science University of Santo Tomas
The Products of Transcription Messenger RNA (mRNA) primary structure linear sequence of RNA bases carries the genetic information in the form of codons
Ribosomal RNA (rRNA)
Codons
assumes a 3D structure
(complexed with proteins) site of protein synthesis
Transfer RNA (tRNA) assumes a cloverleaf structure carries amino acids from cytoplasm to ribosomes
1
Ribosome Encoded by rDNA (ribosomal gene) Synthesized by RNA polymerase I in the nucleolus Complex of RNA and proteins (monosome) Prokaryotes: 10K/cell
Transfer RNA 75-90 nucleotides Nucleotides are post-transcriptionally modified 2D cloverleaf structure (Rodbert Holley) due to base pairing
3D Structure
2D Structure
Amino acid binding site - ends in CCA3’ and 5’G Anticodon loop – contains RNA bases complementary to the codons Other loops serves as recognition sites for enzymes during translation
2
Steps in Translation 1. Charging of tRNA 2. Initiation of translation 3. Elongation of polypeptide chain 4. Termination of translation Charging of tRNA Loading of specific amino acid to its own tRNA Catalyzed by aminoacyl tRNA synthetase 32 different tRNA (despite the presence of 61 codons (bec. Of wobbling mechanisms) 20 different aminoacyl tRNA synthetases Isoaccepting tRNA – tRNA that binds to aa End product: aminoacyl-tRNA complex
Initiation of Translation
Shine-Dalgarmo sequence (5’AGGAGG3’) – sequence that precedes the first codon in prokaryote m RNA Formylmethionine (fmet) – the first amino acid of most polypeptides
3
Elongation of Polypeptide Chain
Peptidyl site (P site) – contains the elongating peptide Aminoacyl site (A site) – contains the amino acid to be added Exit site (E site) – exit of uncharged tRNA Peptidyl transferase – catalyzes the formation of peptide bond High efficiency (error rate 10-4) Rate of elongation: 15 aa/sec at 37°C (E. coli)
Protein Factors Involved in Translation
4
Termination of Translation Signaled by stop codons (UAG, UAA, UGA) Release Factor 1 (RF1) – recognizes stop codon UAA and UAG Release Factor 2 (RF2) – recognizes stop codons UGA and UAA Release factors are GTP dependent Post-translational modification starts after release from ribosome
Polyribosomes Single mRNA being used by different ribosomes for the process of translation Also called polysomes A mechanism to produce more polypeptide (protein) copies
Translation of hemoglobin mRNA in rabbit reticulocyte
Translation in giant salivary gland cells of midgefly
5
Translation in Eukaryotes mRNAs stays in the cytoplasm for longer periods before degradation by RNAses (hours) Ribosomes are much bigger mRNA is capped with 7-methyguanosine (7MG) mRNA contains an initiation sequence called Kozak sequence (ACCAUGG) discovered by Marilyn Kozak Formylmethionine (fMet) is not required for initiation but met is often used as a start codon More complex protein factors involved in different steps Elongating polypeptide enters the ER immediately as translation occurs
Proteins Form Phenotypes Phenyketonuria Mental retardation Autosomal recessive Inability of Phe to converted to Tyr Accumulation of Phe and its derivatives in cerobrospinal fluid
Alkaptonuria Autosomal recessive Darkening ears and nose Benign arthritic conditions
6
Genes and Proteins One gene: one enzyme hypothesis Proposed by George Beadle and Edward Tatum (1940s) Experiments in Neurospora mutants
Genes and Proteins One gene: one protein (polypeptide chain) Not all protein are enzymes Example: Sickle Cell Anemia (mutant hemoglobin)
7
Amino Acids
Protein Structure
Primary Structure
Tertiary Structure
Secondary Structure
Quaternary Structure
8
Post-translational Modifications Cleavage of formylmethionine Cleavage of signal peptides Acetylation of amino group Phosphorylation of certain amino acids Glycosylation Trimmining of polypetides Addition of metallic groups Molecular Chaperons – help proteins undergo correct protein folding to become functional molecules.
Protein Function Structural Function
Collagen Keratin Actin Myosin
Regulatory Function Hormones Hemoglobin Myoglobin
Defense Function Antibodies Complement proteins
Catalytic Function Enzymes Ribozymes
Others Histones Receptors
Enzyme Activity
9
Protein Domains and Exon Shuffling
Structural domains of a fibronnectin molecule
DNA organization of an LDL receptor gene
10
Translation and Proteins John Donnie A. Ramos, Ph.D. Department of Biological Sciences College of Science University of Santo Tomas
The Products of Transcription Messenger RNA (mRNA) primary structure linear sequence of RNA bases carries the genetic information in the form of codons
Ribosomal RNA (rRNA)
Codons
assumes a 3D structure
(complexed with proteins) site of protein synthesis
Transfer RNA (tRNA) assumes a cloverleaf structure carries amino acids from cytoplasm to ribosomes
1
Ribosome Encoded by rDNA (ribosomal gene) Synthesized by RNA polymerase I in the nucleolus Complex of RNA and proteins (monosome) Prokaryotes: 10K/cell
Transfer RNA 75-90 nucleotides Nucleotides are post-transcriptionally modified 2D cloverleaf structure (Rodbert Holley) due to base pairing
3D Structure
2D Structure
Amino acid binding site - ends in CCA3’ and 5’G Anticodon loop – contains RNA bases complementary to the codons Other loops serves as recognition sites for enzymes during translation
2
Steps in Translation 1. Charging of tRNA 2. Initiation of translation 3. Elongation of polypeptide chain 4. Termination of translation Charging of tRNA Loading of specific amino acid to its own tRNA Catalyzed by aminoacyl tRNA synthetase 32 different tRNA (despite the presence of 61 codons (bec. Of wobbling mechanisms) 20 different aminoacyl tRNA synthetases Isoaccepting tRNA – tRNA that binds to aa End product: aminoacyl-tRNA complex
Initiation of Translation
Shine-Dalgarmo sequence (5’AGGAGG3’) – sequence that precedes the first codon in prokaryote m RNA Formylmethionine (fmet) – the first amino acid of most polypeptides
3
Elongation of Polypeptide Chain
Peptidyl site (P site) – contains the elongating peptide Aminoacyl site (A site) – contains the amino acid to be added Exit site (E site) – exit of uncharged tRNA Peptidyl transferase – catalyzes the formation of peptide bond High efficiency (error rate 10-4) Rate of elongation: 15 aa/sec at 37°C (E. coli)
Protein Factors Involved in Translation
4
Termination of Translation Signaled by stop codons (UAG, UAA, UGA) Release Factor 1 (RF1) – recognizes stop codon UAA and UAG Release Factor 2 (RF2) – recognizes stop codons UGA and UAA Release factors are GTP dependent Post-translational modification starts after release from ribosome
Polyribosomes Single mRNA being used by different ribosomes for the process of translation Also called polysomes A mechanism to produce more polypeptide (protein) copies
Translation of hemoglobin mRNA in rabbit reticulocyte
Translation in giant salivary gland cells of midgefly
5
Translation in Eukaryotes mRNAs stays in the cytoplasm for longer periods before degradation by RNAses (hours) Ribosomes are much bigger mRNA is capped with 7-methyguanosine (7MG) mRNA contains an initiation sequence called Kozak sequence (ACCAUGG) discovered by Marilyn Kozak Formylmethionine (fMet) is not required for initiation but met is often used as a start codon More complex protein factors involved in different steps Elongating polypeptide enters the ER immediately as translation occurs
Proteins Form Phenotypes Phenyketonuria Mental retardation Autosomal recessive Inability of Phe to converted to Tyr Accumulation of Phe and its derivatives in cerobrospinal fluid
Alkaptonuria Autosomal recessive Darkening ears and nose Benign arthritic conditions
6
Genes and Proteins One gene: one enzyme hypothesis Proposed by George Beadle and Edward Tatum (1940s) Experiments in Neurospora mutants
Genes and Proteins One gene: one protein (polypeptide chain) Not all protein are enzymes Example: Sickle Cell Anemia (mutant hemoglobin)
7
Amino Acids
Protein Structure
Primary Structure
Tertiary Structure
Secondary Structure
Quaternary Structure
8
Post-translational Modifications Cleavage of formylmethionine Cleavage of signal peptides Acetylation of amino group Phosphorylation of certain amino acids Glycosylation Trimmining of polypetides Addition of metallic groups Molecular Chaperons – help proteins undergo correct protein folding to become functional molecules.
Protein Function Structural Function
Collagen Keratin Actin Myosin
Regulatory Function Hormones Hemoglobin Myoglobin
Defense Function Antibodies Complement proteins
Catalytic Function Enzymes Ribozymes
Others Histones Receptors
Enzyme Activity
9
Protein Domains and Exon Shuffling
Structural domains of a fibronnectin molecule
DNA organization of an LDL receptor gene
10
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