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What is a gene?

What is a gene?. A sequence of DNA nucleotides that specifies the primary structure of a polypeptide chain (tells the cell how to make it) Genes-made of nucleotides Proteins-made of amino acids How does a nucleotide code (in the nucleus) specify an amino acid sequence (in the cytoplasm)?.

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What is a gene?

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  1. What is a gene? • A sequence of DNA nucleotides that specifies the primary structure of a polypeptide chain (tells the cell how to make it) • Genes-made of nucleotides • Proteins-made of amino acids • How does a nucleotide code (in the nucleus) specify an amino acid sequence (in the cytoplasm)?

  2. The Central Dogma • DNA is transcribed into RNA-characteristics of RNA • RNA is translated into protein • Advantages • Exceptions

  3. Gene 2 DNA molecule Gene 1 Gene 3 LE 17-4 5¢ 3¢ DNA strand (template) TRANSCRIPTION 5¢ 3¢ mRNA Codon TRANSLATION Protein Amino acid

  4. The Genetic code-characteristics • Triplet (3 nucleotides=codon=info for a specific amino acid);64 different codons (3 are stop codons) • Universal • Redundant (61 codons-20 amino acids)-variability in third nucleotide of codon. Advantages of a redundant code? • Non-overlapping • Exceptions (ciliates; mito/chloroplasts)

  5. Second mRNA base LE 17-5 First mRNA base (5¢ end) Third mRNA base (3¢ end)

  6. Figure 17-06

  7. Gene Expression • If a gene is transcribed and the m-rna is translated (the gene is expressed); a protein is made. This often changes the phenotype of the cell that produces the protein. • Differential gene expression is involved in embryonic development and cell specialization. • Totipotency-each cell has the genetic information for an entire organism. • Differential gene expression results in cell specialization (differentiation) • Hormones often play a role in gene expression

  8. Transcription • The first step in gene expression • Takes place in the nucleus • Requirements • A. RNA nucleotides • B. DNA template (gene) • C. Enzymes (RNA polymerase) • Only one of the two DNA strands is copied (template strand)

  9. Transcription unit Promoter LE 17-7a-1 5¢ 3¢ 3¢ 5¢ DNA Start point RNA polymerase

  10. Promoter Transcription unit 5¢ 3¢ 5¢ 3¢ DNA Start point LE 17-7a-2 RNA polymerase Initiation 5¢ 3¢ 3¢ 5¢ Template strand of DNA RNA tran- script Unwound DNA

  11. Promoter Transcription unit 5¢ 3¢ 5 3¢ DNA Start point RNA polymerase Initiation LE 17-7a-3 5¢ 3¢ 5 3¢ Template strand of DNA RNA tran- script Unwound DNA Elongation Rewound DNA 5¢ 3¢ 3¢ 5 3¢ 5¢ RNA transcript

  12. Promoter Transcription unit 5 3 3¢ 5¢ DNA Start point RNA polymerase Initiation LE 17-7a-4 5¢ 3¢ 5¢ 3¢ Template strand of DNA RNA tran- script Unwound DNA Elongation Rewound DNA 5¢ 3¢ 3¢ 3¢ 5¢ 5¢ RNA transcript Termination 5¢ 3¢ 3¢ 5¢ 5¢ 3¢ Completed RNA transcript

  13. Elongation Non-template strand of DNA RNA nucleotides RNA polymerase LE 17-7b 3¢ 3¢ end 5¢ Direction of transcription (“downstream”) 5¢ Template strand of DNA Newly made RNA

  14. Eukaryotic promoters Promoter 5¢ 3¢ 3¢ 5¢ TATA box Start point Template DNA strand Several transcription factors Transcription factors LE 17-8 5¢ 3¢ 3¢ 5¢ Additional transcription factors RNA polymerase II Transcription factors 5¢ 3¢ 5¢ 3¢ 5¢ RNA transcript Transcription initiation complex

  15. Transcription-some important details • Rate-30-60 nucleotides/second • RNA polymerase (Many forms in eucaryotes, 3 basic types in bacteria: type I transcribes r-rna, type II-mrna, types III-trna) • Promotors-(approximately 100 nucleotides)-strong and weak promotors • Eukaryotes-transcription factors needed to help RNA polymerase to bind to TATA box (region of promotor 25 nucleotides upstream from initiation site).

  16. RNA products of transcription

  17. Recent discoveries indicate that a large part of the eukaryotic genome is non-coding RNA • R-rna and T-trna are examples • Small rna (micro rna and small interfering rna)-play a crucial role in the regulation of gene expression involving both transcription and translation • Rna interference • We’ll talk about regulation of gene expression in Chapter 18.

  18. Ribosomal RNA and ribosomes • R-rna; one of two important components of ribosomes (other is protein-some of the proteins are enzymes). 60% r-rna; 40% protein. • Ribosomes consist of 2 subunits • Ribosomes needed to translate proteins • “workbench of protein synthesis” • Position t-rna (which is attached to a specific amino acid) on the codon of a m-rna • Result is the synthesis of a protein (whose amino acid sequence is specified by the m-rna; which is transcribed from a gene)

  19. P site (Peptidyl-tRNA binding site) LE 17-16b A site (Aminoacyl- tRNA binding site) E site (Exit site) E P A Large subunit mRNA binding site Small subunit Schematic model showing binding sites

  20. Exit tunnel Growing polypeptide tRNA molecules LE 17-16a Large subunit E P A Small subunit 5¢ 3¢ mRNA Computer model of functioning ribosome

  21. Ribosomal –rna processing

  22. T-rna • Single polynucleotide chain folded into a complex 3-D shape (inter-chain H bonding). 75-80 nucleotides in length • Binds a specific amino acid (involvement of amino-acyl-trna-synthetase • Attaches to a specific m-rna codon via its anticodon • How many different t-rna’s are there? 61? Actually only 45 (wobble)

  23. 3¢ Amino acid attachment site 5¢ LE 17-14a Hydrogen bonds Anticodon Two-dimensional structure Amino acid attachment site 5¢ 3¢ Hydrogen bonds 3¢ 5¢ Anticodon Anticodon Three-dimensional structure Symbol used in this book

  24. “Charging” t-rna with its specific amino acid • “charging” enzyme-amino acyl t-rna synthetase (20 different enzymes) • Requires ATP

  25. Amino acid Aminoacyl-tRNA synthetase (enzyme) LE 17-15 Pyrophosphate Phosphates tRNA AMP Aminoacyl tRNA (an “activated amino acid”)

  26. Messenger Rna (m-rna) • Contains the information for the primary sequence of a polypeptide chain • Consists of codons • Binds to ribosomes • T-rna binds to m-rna (codon/anticodon)

  27. Amino acids LE 17-13 Polypeptide tRNA with amino acid attached Ribosome tRNA Anticodon Codons 5¢ 3¢ mRNA

  28. Translation • Codons (m-rna) read by ribosomes/t-rna • Polypeptide chain produced • 3 steps in translation- • A. initiation • B. elongation • C. termination • Translation is a process that consumes a tremendous amount of energy (ATP and GTP)

  29. Amino end Growing polypeptide LE 17-16c Next amino acid to be added to polypeptide chain E tRNA mRNA 3¢ Codons 5¢ Schematic model with mRNA and tRNA

  30. Translation-Initiation • Initiation codon is AUG • T-rna that bonds to AUG has an anticodon UAC-this carries the amino acid methionine • Requires a GTP molecule • Requires proteins called initiation factors.

  31. Large ribosomal subunit LE 17-17 P site Met Met Initiator tRNA GTP GDP A E mRNA 5¢ 5¢ 3¢ 3¢ Start codon Small ribosomal subunit mRNA binding site Translation initiation complex

  32. Translation-Elongation • The elongation cycle takes about 60 milliseconds • During elongation, one m-rna codon is read and then the ribosomes moves down the message to the next codon. • Binding of incoming t-rna to the A site of the ribosome requires a GTP • Translocation-requires a GTP

  33. Amino end of polypeptide E 3¢ mRNA P site A site Ribosome ready for next aminoacyl tRNA 5¢ GTP LE 17-18 2 2 GDP E E P A P A GDP GTP E P A

  34. Translation-Termination • When the ribosome reaches a termination codon, it causes the m-rna/ribosome complex to separate • No t-rna binds to the termination codon. • Release factors • Newly made polypeptide chain is released (folds into its characteristic 3-D shape)

  35. LE 17-19 Release factor Free polypeptide 5¢ 3¢ 3¢ 3¢ 5¢ 5¢ Stop codon (UAG, UAA, or UGA) When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. The two ribosomal subunits and the other components of the assembly dissociate.

  36. Summary of energy demands for protein synthesis • A rough estimate is that for every amino acid incorporated into a polypeptide chain, 3 ATP/GTP are consumed • Charging the amino acid (1 ATP) • Binding of incoming t-rna into the A site (1 GTP) • Translocation (1 GTP) • So a small protein (120 amino acids in length) would cost the cell 360 ATP/GTP to make (the equivalent of 12 glucose molecules going through aerobic cell respiration)

  37. Polyribosomes • A single ribosome can translate an average-sized polypeptide in about 1 minute • Several ribosomes can translate the same message one after the other. • Increases the efficiency of protein production

  38. Completed polypeptide Growing polypeptides LE 17-20a Incoming ribosomal subunits Polyribosome Start of mRNA (5¢ end) End of mRNA (3¢ end) An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes.

  39. Ribosomes LE 17-20b mRNA m 0.1 m This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

  40. M-rna modifications • Eukaryotic M-rna is modified extensively after transcription (while its still in the nucleus) • These modifications include A.Polyadenylation-added to 3’ end of m-rna B. 5’ cap C. Intron removal

  41. M-RNA modifications • Poly A tail • A. added to the 3’ end of the m-rna • B.30-200 Adenine nucleotides • C. roles-regulation of transport of m-rna out of the nucleus; regulation of degradation of m-rna in the cytoplasm; helps m-rna attach to small ribosomal subunit

  42. M-RNA modifications (continued) • 5’ cap • A. Modified guanine nucleotide stuck onto 5’ end of m-rna • B. Roles- positioning of m-rna on small ribosomal subunit in initiation; protects m-rna from degradation

  43. LE 17-9 Protein-coding segment Polyadenylation signal 5¢ Start codon Stop codon 3¢ UTR Cap 5¢ UTR 5¢ Poly-A tail

  44. Introns • Many eukaryotic genes have nucleotide sequences that don’t code for amino acids (Introns) • Introns separate coding sequences (exons). Split genes • Introns must be removed from the m-rna before it is translated (introns have nucleotide sequences that indicate splicing sites) • Splicesomes are molecular machines that remove introns from m-rna

  45. RNA transcript (pre-mRNA) 5¢ Intron Exon 1 Exon 2 LE 17-11-1 Protein Other proteins snRNA snRNPs Spliceosome

  46. Spliceosome 5¢ LE 17-11-2 Spliceosome components Cut-out intron mRNA 5¢ Exon 2 Exon 1

  47. Significance of introns • Why would chromosomes carry around extra DNA that isn’t used in the final m-rna? • Expensive to maintain (energy). • Splicing out introns is a risky business (what if it’s done incorrectly) • With these disadvantages, there must be an advantage or natural selection would not favor this arrangement

  48. Benefits of Introns • Evolution of protein diversity • One gene can be alternatively spliced in a number of different ways to form several different types of m-rna (alternative splicing) • Human antibody genes-about 500 genes can code for billions of different antibody molecules because of alternative splicing.

  49. Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription LE 17-12 RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide

  50. Summary of Transcription and Translation

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