14 Gene Expression: From Gene to Protein

Transcription

1 CMPBELL BIOLOY IN FOCS rry Cain Wasserman Minorsky Jackson Reece 14 ene Expression: From ene to Protein Lecture Presentations by Kathleen Fitzpatrick and Nicole Tunbridge

2 Overview: The Flow of enetic Information The information content of genes is in the form of specific sequences of nucleotides in DN The DN inherited by an organism leads to specific traits by dictating the synthesis of proteins Proteins are the links between genotype and phenotype ene expression, the process by which DN directs protein synthesis, includes two stages: transcription and translation

3 Figure 14.1

4 Concept 14.1: enes specify proteins via transcription and translation How was the fundamental relationship between genes and proteins discovered?

5 Basic Principles of Transcription and Translation RN is the bridge between DN and protein synthesis RN is chemically similar to DN, but RN has a ribose sugar and the base uracil () rather than thymine (T) RN is usually single-stranded etting from DN to protein requires two stages: transcription and translation

6 Transcription is the synthesis of RN using information in DN Transcription produces messenger RN (mrn) Translation is the synthesis of a polypeptide, using information in the mrn Ribosomes are the sites of translation

7 In prokaryotes, translation of mrn can begin before transcription has finished In eukaryotes, the nuclear envelope separates transcription from translation Eukaryotic RN transcripts are modified through RN processing to yield the finished mrn Eukaryotic mrn must be transported out of the nucleus to be translated

8 Figure 14.4 Nuclear envelope TRNSCRIPTION DN RN PROCESSIN Pre-mRN TRNSCRIPTION DN mrn TRNSLTION mrn Ribosome TRNSLTION Ribosome Polypeptide Polypeptide (a) Bacterial cell (b) Eukaryotic cell

9 primary transcript is the initial RN transcript from any gene prior to processing The central dogma is the concept that cells are governed by a cellular chain of command

10 Figure 14.N01 DN RN Protein

11 The enetic Code How are the instructions for assembling amino acids into proteins encoded into DN? There are 20 amino acids, but there are only four nucleotide bases in DN How many nucleotides correspond to an amino acid?

12 Codons: Triplets of Nucleotides The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping, three-nucleotide words The words of a gene are transcribed into complementary nonoverlapping three-nucleotide words of mrn These words are then translated into a chain of amino acids, forming a polypeptide

13 Figure 14.5 DN template strand C C C C T T T T T C T C TRNSCRIPTION mrn C C TRNSLTION Codon Protein Trp Phe ly Ser mino acid

14 During transcription, one of the two DN strands, called the template strand, provides a template for ordering the sequence of complementary nucleotides in an RN transcript The template strand is always the same strand for any given gene

15 During translation, the mrn base triplets, called codons, are read in the to direction Each codon specifies the amino acid (one of 20) to be placed at the corresponding position along a polypeptide

16 Cracking the Code ll 64 codons were deciphered by the mid-1960s Of the 64 triplets, 61 code for amino acids; 3 triplets are stop signals to end translation The genetic code is redundant: more than one codon may specify a particular amino acid But it is not ambiguous: no codon specifies more than one amino acid

17 Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced Codons are read one at a time in a nonoverlapping fashion

18 First mrn base ( end of codon) Third mrn base ( end of codon) Figure 14.6 Second mrn base C C Phe Leu C CC C C Ser C Tyr Stop Stop C Cys Stop Trp C C C CC C C Leu CC CCC CC CC Pro C CC C C His ln C CC C C rg C C IIe Met or start C CC C C Thr C sn Lys C Ser rg C C Val C CC C C la C sp lu C ly C

19 Evolution of the enetic Code The genetic code is nearly universal, shared by the simplest bacteria and the most complex animals enes can be transcribed and translated after being transplanted from one species to another

20 Figure 14.7 (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene

21 Concept 14.2: Transcription is the DN-directed synthesis of RN: a closer look Transcription is the first stage of gene expression

22 Molecular Components of Transcription RN synthesis is catalyzed by RN polymerase, which pries the DN strands apart and joins together the RN nucleotides RN polymerases assemble polynucleotides in the to direction However, RN polymerases can start a chain without a primer

23 Figure Promoter Transcription unit 1 Initiation Start point RN polymerase 2 3 Elongation Termination nwound DN Rewound DN RN transcript RN Template strand of DN transcript Completed RN transcript Direction of transcription ( downstream )

24 The DN sequence where RN polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator The stretch of DN that is transcribed is called a transcription unit

25 Synthesis of an RN Transcript The three stages of transcription Initiation Elongation Termination

26 RN Polymerase Binding and Initiation of Transcription Promoters signal the transcriptional start point and usually extend several dozen nucleotide pairs upstream of the start point Transcription factors mediate the binding of RN polymerase and the initiation of transcription

27 The completed assembly of transcription factors and RN polymerase II bound to a promoter is called a transcription initiation complex promoter called a TT box is crucial in forming the initiation complex in eukaryotes

28 Figure 14.N02 TRNSCRIPTION DN RN PROCESSIN Pre-mRN mrn TRNSLTION Ribosome Polypeptide

29 Figure 14.9 DN T T T T T T T TT box Promoter Transcription factors Start point Nontemplate strand Template strand 1 eukaryotic promoter 2 Several transcription factors bind to DN. RN polymerase II Transcription factors 3 Transcription initiation complex forms. RN transcript Transcription initiation complex

30 Elongation of the RN Strand s RN polymerase moves along the DN, it untwists the double helix, 10 to 20 bases at a time Transcription progresses at a rate of 40 nucleotides per second in eukaryotes gene can be transcribed simultaneously by several RN polymerases

31 Figure RN polymerase Nontemplate strand of DN RN nucleotides T C C end C C T T T Newly made RN Direction of transcription Template strand of DN

32 Termination of Transcription The mechanisms of termination are different in bacteria and eukaryotes In bacteria, the polymerase stops transcription at the end of the terminator and the mrn can be translated without further modification In eukaryotes, RN polymerase II transcribes the polyadenylation signal sequence; the RN transcript is released nucleotides past this polyadenylation sequence

33 Concept 14.3: Eukaryotic cells modify RN after transcription Enzymes in the eukaryotic nucleus modify premrn (RN processing) before the genetic messages are dispatched to the cytoplasm During RN processing, both ends of the primary transcript are altered lso, usually some interior parts of the molecule are cut out and the other parts spliced together

34 lteration of mrn Ends Each end of a pre-mrn molecule is modified in a particular way The end receives a modified nucleotide cap The end gets a poly- tail These modifications share several functions Facilitating the export of mrn to the cytoplasm Protecting mrn from hydrolytic enzymes Helping ribosomes attach to the end

35 Figure 14.N03 TRNSCRIPTION DN RN PROCESSIN Pre-mRN mrn TRNSLTION Ribosome Polypeptide

36 Figure modified guanine nucleotide added to the end P Protein-coding segment P P adenine nucleotides added to the end Polyadenylation signal Start Stop Cap TR TR Poly- tail codon codon

37 Split enes and RN Splicing Most eukaryotic mrns have long noncoding stretches of nucleotides that lie between coding regions The noncoding regions are called intervening sequences, or introns The other regions are called exons and are usually translated into amino acid sequences RN splicing removes introns and joins exons, creating an mrn molecule with a continuous coding sequence

38 Figure Pre-mRN Cap Intron mrn Intron Introns cut out and exons spliced together Poly- tail Cap TR Coding segment Poly- tail TR

39 Many genes can give rise to two or more different polypeptides, depending on which segments are used as exons This process is called alternative RN splicing

40 Concept 14.4: Translation is the RN-directed synthesis of a polypeptide: a closer look enetic information flows from mrn to protein through the process of translation

41 Molecular Components of Translation cell translates an mrn message into protein with the help of transfer RN (trn) trns transfer amino acids to the growing polypeptide in a ribosome Translation is a complex process in terms of its biochemistry and mechanics

42 Figure 14.N04 TRNSCRIPTION DN TRNSLTION mrn Polypeptide Ribosome

43 Figure Polypeptide mino acids Ribosome trn with amino acid attached ly C trn nticodon mrn Codons

44 The Structure and Function of Transfer RN Each trn can translate a particular mrn codon into a given amino acid The trn contains an amino acid at one end and at the other end has a nucleotide triplet that can basepair with the complementary codon on mrn

45 trn molecule consists of a single RN strand that is only about 80 nucleotides long trn molecules can base-pair with themselves Flattened into one plane, a trn molecule looks like a cloverleaf In three dimensions, trn is roughly L-shaped, where one end of the L contains the anticodon that base-pairs with an mrn codon

46 Figure mino acid attachment site * C C C C C C C C C * C C * C * * C * * C C * * C * nticodon * * Hydrogen bonds (a) Two-dimensional structure Hydrogen bonds nticodon (b) Three-dimensional structure mino acid attachment site nticodon (c) Symbol used in this book

47 Ribosomes Ribosomes facilitate specific coupling of trn anticodons with mrn codons during protein synthesis The large and small ribosomal are made of proteins and ribosomal RNs (rrns) In bacterial and eukaryotic ribosomes the large and small subunits join to form a ribosome only when attached to an mrn molecule

48 Figure rowing polypeptide trn molecules Exit tunnel E P Large subunit Small subunit mrn (a) Computer model of functioning ribosome P site (Peptidyl-tRN binding site) E site (Exit site) E P Exit tunnel site (minoacyltrn binding site) Large subunit mrn mino end E rowing polypeptide Next amino acid to be added to polypeptide chain trn mrn binding site Small subunit (b) Schematic model showing binding sites Codons (c) Schematic model with mrn and trn

49 ribosome has three binding sites for trn The P site holds the trn that carries the growing polypeptide chain The site holds the trn that carries the next amino acid to be added to the chain The E site is the exit site, where discharged trns leave the ribosome

50 Building a Polypeptide The three stages of translation Initiation Elongation Termination ll three stages require protein factors that aid in the translation process

51 Ribosome ssociation and Initiation of Translation The initiation stage of translation brings together mrn, a trn with the first amino acid, and the two ribosomal subunits small ribosomal subunit binds with mrn and a special initiator trn Then the small subunit moves along the mrn until it reaches the start codon ()

52 Figure C P site Large ribosomal subunit Initiator trn mrn TP Start codon Small mrn binding site ribosomal subunit 1 Small ribosomal subunit binds 2 to mrn. P i DP E Translation initiation complex Large ribosomal subunit completes the initiation complex.

53 The start codon is important because it establishes the reading frame for the mrn The addition of the large ribosomal subunit is last and completes the formation of the translation initiation complex Proteins called initiation factors bring all these components together

54 Elongation of the Polypeptide Chain During elongation, amino acids are added one by one to the previous amino acid at the C-terminus of the growing chain Each addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bond formation, and translocation Translation proceeds along the mrn in a to direction

55 Figure mino end of polypeptide 1 Codon recognition mrn E P site site TP DP P i E P

56 Figure mino end of polypeptide 1 Codon recognition mrn E P site site TP DP P i E P 2 Peptide bond formation E P

57 Figure mino end of polypeptide 1 Codon recognition Ribosome ready for next aminoacyl trn mrn E P site site TP DP P i E E P P 3 Translocation DP P i TP 2 Peptide bond formation E P

58 Termination of Translation Termination occurs when a stop codon in the mrn reaches the site of the ribosome The site accepts a protein called a release factor The release factor causes the addition of a water molecule instead of an amino acid This reaction releases the polypeptide, and the translation assembly then comes apart

59 Figure Release factor 1 Stop codon (,, or ) Ribosome reaches a stop codon on mrn.

60 Figure Release factor Free polypeptide Stop codon (,, or ) 1 Ribosome reaches a stop codon on mrn. 2 Release factor promotes hydrolysis.

61 Figure Release factor Free polypeptide 1 Stop codon (,, or ) Ribosome reaches a stop codon on mrn. 2 TP 2 Release factor 3 promotes hydrolysis. 2 DP P i Ribosomal subunits and other components dissociate.

62 Figure TRNSCRIPTION DN RN transcript RN PROCESSIN CYTOPLSM Exon NCLES RN polymerase RN transcript (pre-mrn) Intron mino acid trn minoacyl-trn synthetase MINO CID CTIVTION mrn E P Ribosomal subunits minoacyl (charged) trn TRNSLTION E nticodon Ribosome Codon

63 Figure 14.24a TRNSCRIPTION DN RN transcript RN PROCESSIN CYTOPLSM Exon NCLES RN polymerase RN transcript (pre-mrn) Intron mino acid trn minoacyl-trn synthetase MINO CID CTIVTION mrn minoacyl (charged) trn

64 Figure 14.24b mrn rowing polypeptide E P Ribosomal subunits minoacyl (charged) trn TRNSLTION E nticodon Ribosome Codon

65 Concept 14.5: Mutations of one or a few nucleotides can affect protein structure and function Mutations are changes in the genetic material of a cell or virus Point mutations are chemical changes in just one or a few nucleotide pairs of a gene The change of a single nucleotide in a DN template strand can lead to the production of an abnormal protein

66 Figure Wild-type hemoglobin Sickle-cell hemoglobin Wild-type hemoglobin DN C T C Mutant hemoglobin DN C C T mrn mrn Normal hemoglobin lu Sickle-cell hemoglobin Val

67 Types of Small-Scale Mutations Point mutations within a gene can be divided into two general categories Nucleotide-pair substitutions One or more nucleotide-pair insertions or deletions

68 Figure T T Met DN template strand T mrn Protein mino end (a) Nucleotide-pair substitution Lys Phe ly C T T C C C T T T T T T C T Met C T T C C C T T T T T T T T Met Lys Phe Ser Stop Silent (no effect on amino acid sequence) Missense C T T C T C T T T T T T C T T C T C T instead of Met Nonsense instead of T C C T T T T T T C T Stop instead of instead of C T instead of C instead of C Stop Wild type Lys Phe T T T ly Met C C Stop Carboxyl end (b) Nucleotide-pair insertion or deletion Extra T T C C C T T T T T T C T C T T C C C T T T T T C T Met T Met Extra T T C C Lys Phe Leu C C ly la T T T T C T C T Stop Frameshift causing immediate nonsense (1 nucleotide-pair insertion) Frameshift causing extensive missense (1 nucleotide-pair deletion) missing missing missing missing Stop No frameshift, but one amino acid missing (3 nucleotide-pair deletion) T

69 Substitutions nucleotide-pair substitution replaces one nucleotide and its partner with another pair of nucleotides Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code

70 Figure 14.26a Wild type DN template strand mrn Protein mino end T C T T C C C T T T T T T C T Met Lys Phe C ly Stop Carboxyl end Nucleotide-pair substitution: silent instead of T C T T C C C T T T T T T T T Met Lys Phe ly instead of C Stop

71 Missense mutations still code for an amino acid, but not the correct amino acid Substitution mutations are usually missense mutations Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

72 Figure 14.26b Wild type DN template strand mrn Protein mino end T C T T C C C T T T T T T C T Met Lys Phe C ly Stop Carboxyl end Nucleotide-pair substitution: missense Met Lys T instead of C T C T T C T C T T T T T T C T instead of Phe C Ser Stop

73 Figure 14.26c Wild type DN template strand mrn Protein mino end T C T T C C C T T T Met Lys T T Phe T C C ly T Stop Carboxyl end Nucleotide-pair substitution: nonsense instead of T T C T C C C T T T T T T T C T instead of Met Stop C

74 Insertions and Deletions Insertions and deletions are additions or losses of nucleotide pairs in a gene These mutations have a disastrous effect on the resulting protein more often than substitutions do Insertion or deletion of nucleotides may alter the reading frame of the genetic message, producing a frameshift mutation

75 Figure 14.26d Wild type DN template strand mrn Protein mino end T C T T C C C T T T T T T C T Met Lys Phe C ly Stop Carboxyl end Nucleotide-pair insertion: frameshift causing immediate nonsense Extra T C T T C C C T T T Met T Extra Stop T T T C T C

76 Figure 14.26e Wild type DN template strand mrn Protein mino end T C T T C C C T T T Met Lys T T Phe T C C ly T Stop Carboxyl end Nucleotide-pair deletion: frameshift causing extensive missense missing T C T T C C C T T T T T C T missing C Met Lys Leu la

77 Figure 14.26f Wild type DN template strand mrn Protein mino end T C T T C C C T T T Met Lys T T Phe T C C ly T Stop Carboxyl end 3 nucleotide-pair deletion: no frameshift, but one amino acid missing T T C missing T C C C T T T T T T C T missing C Met Phe ly Stop

78 Mutagens Spontaneous mutations can occur during DN replication, recombination, or repair Mutagens are physical or chemical agents that can cause mutations Researchers have developed methods to test the mutagenic activity of chemicals Most cancer-causing chemicals (carcinogens) are mutagenic, and the converse is also true