Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The Flow of Genetic Information The information content of DNA is in the form of specific sequences of nucleotides The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation The ribosome is part of the cellular machinery for translation, polypeptide synthesis

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Genes specify proteins via transcription and translation Transcription is the synthesis of RNA under the direction of DNA Transcription produces messenger RNA (mRNA) Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA Ribosomes are the sites of translation

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings In prokaryotes, mRNA produced by transcription is immediately translated without more processing In a eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA

LE TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide DNA Pre-mRNA Prokaryotic cell Nuclear envelope mRNA TRANSLATION TRANSCRIPTION RNA PROCESSING Ribosome Polypeptide Eukaryotic cell

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The Genetic Code How are the instructions for assembling amino acids into proteins encoded into DNA? There are 20 amino acids, but there are only four nucleotide bases in DNA So how many bases correspond to an amino acid?

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Codons: Triplets of Bases The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping, three-nucleotide words These triplets are the smallest units of uniform length that can code for all the amino acids Example: AGT at a particular position on a DNA strand results in the placement of the amino acid serine at the corresponding position of the polypeptide to be produced

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings During transcription, a DNA strand called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript During translation, the mRNA base triplets, called codons, are read in the 5 to 3 direction Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide

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

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Evolution of the Genetic Code The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals Genes can be transcribed and translated after being transplanted from one species to another

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Transcription is the DNA-directed synthesis of RNA: a closer look RNA synthesis is catalyzed by RNA polymerase, which opens the DNA double helix and synthesizes an RNA copy RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine The DNA sequence where RNA polymerase attaches is called the promoter; in prokaryotes, the sequence signaling the end of transcription is called the terminator

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

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Synthesis of an RNA Transcript The three stages of transcription: – Initiation – Elongation – Termination

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings RNA Polymerase Binding and Initiation of Transcription Promoters signal the initiation of RNA synthesis Transcription factors mediate the binding of RNA polymerase and initiation of transcription The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Elongation of the RNA Strand As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time Transcription progresses at a rate of 60 nucleotides per second in eukaryotes A gene can be transcribed simultaneously by several RNA polymerases

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Termination of Transcription The mechanisms of termination are different in prokaryotes and eukaryotes In prokaryotes, the polymerase stops transcription at the end of the terminator In eukaryotes, the polymerase continues transcription after the pre-mRNA is cleaved from the growing RNA chain; the polymerase eventually falls off the DNA

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Eukaryotic cells modify RNA after transcription Enzymes in the eukaryotic nucleus modify pre- mRNA before the genetic messages are dispatched to the cytoplasm

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Alteration of mRNA Ends Each end of a pre-mRNA molecule is modified in a particular way: – The 5 end receives a modified nucleotide cap – The 3 end receives a poly-A tail These modifications share several functions: – They seem to facilitate export from the nucleus – They protect mRNA from hydrolytic enzymes – They help ribosomes attach to the 5’ end

LE Protein-coding segment 5 Start codon Stop codon Poly-A tail Polyadenylation signal 5 3 Cap UTR

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Removal of Introns - RNA Splicing Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions These noncoding regions are called intervening sequences, or introns The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

LE ExonIntronExonIntronExon 3 Pre-mRNA Coding segment Introns cut out and exons spliced together  Cap Poly-A tail 5 3 UTR

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings In some cases, RNA splicing is carried out by spliceosomes Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites

LE Exon 1 5 IntronExon 2 Other proteins Protein snRNA snRNPs RNA transcript (pre-mRNA) Spliceosome 5 components Cut-out intron mRNA Exon 1Exon 2 5

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The Functional and Evolutionary Importance of Introns Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing Such variations are called alternative RNA splicing Because of alternative splicing, the number of different proteins an organism can produce is much greater than its number of genes

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Proteins often have a modular architecture consisting of discrete regions called domains In many cases, different exons code for the different domains in a protein

LE Gene Transcription RNA processing Translation Domain 2 Domain 3 Domain 1 Polypeptide Exon 1IntronExon 2IntronExon 3 DNA

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Translation is the RNA-directed synthesis of a polypeptide: a closer look A cell translates an mRNA message into protein with the help of transfer RNA (tRNA) Molecules of tRNA are not identical: – Each carries a specific amino acid on one end – Each has an anticodon on the other end; the anticodon base-pairs with a complementary codon on mRNA

LE Polypeptide tRNA with amino acid attached Ribosome tRNA Anticodon 3 5 mRNA Amino acids Codons

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The Structure and Function of Transfer RNA A C C A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long Some parts base pair to make stems. Other parts make loops. Because of hydrogen bonds, tRNA actually twists and folds into a three-dimensional molecule tRNA is roughly L-shaped

LE 17-14a Amino acid attachment site Hydrogen bonds 3 5 Two-dimensional structure Anticodon Amino acid attachment site 3 5 Hydrogen bonds Anticodon Symbol used in this book Three-dimensional structure 35

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Accurate translation requires two steps: – First step: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase – Second step: a correct match between the tRNA anticodon and an mRNA codon

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Ribosomes Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings A ribosome has three binding sites for tRNA: – The A site holds the tRNA that carries the next amino acid to be added to the chain – The P site holds the tRNA that carries the growing polypeptide chain – The E site is the exit site, where discharged tRNAs leave the ribosome

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Building a Polypeptide The three stages of translation: – Initiation – Elongation – Termination All three stages require protein “factors” that aid in the translation process

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Ribosome Association and Initiation of Translation The initiation stage of translation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits First, a small ribosomal subunit binds with mRNA and a special initiator tRNA Then the small subunit moves along the mRNA until it reaches the start codon (AUG) Proteins called initiation factors bring in the large subunit so the initiator tRNA occupies the P site

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

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Elongation of the Polypeptide Chain During the elongation stage, amino acids are added one by one to the preceding amino acid Each addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bond formation, and translocation

LE Ribosome ready for next aminoacyl tRNA mRNA 5 Amino end of polypeptide E P site A site GDP E PA GTP GDP E PA E PA

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Termination of Translation Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome The A site accepts a protein called a release factor The release factor causes the addition of a water molecule instead of an amino acid This releases the polypeptide, and the translation assembly then comes apart

LE 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. Release factor Stop codon (UAG, UAA, or UGA) Free polypeptide 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.

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Polyribosomes A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome Polyribosomes enable a cell to make many copies of a polypeptide very quickly

LE Ribosomes mRNA 0.1  m This micrograph shows a large polyribosome in a prokaryotic cell (TEM). An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Incoming ribosomal subunits Growing polypeptides End of mRNA (3 end) Start of mRNA (5 end) Polyribosome Completed polypeptides

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Completing and Targeting the Functional Protein Often translation is not sufficient to make a functional protein Polypeptide chains are modified after translation Completed proteins are targeted to specific sites in the cell

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Protein Folding and Post-Translational Modifications During and after synthesis, a polypeptide chain spontaneously coils and folds into its three- dimensional shape This is sometimes aided by chaperone proteins Proteins may also require posttranslational modifications before doing their job

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Targeting Polypeptides to Specific Locations Two populations of ribosomes are evident in cells: free ribsomes (in the cytosol) and bound ribosomes (attached to the ER) Free ribosomes mostly synthesize proteins that function in the cytosol Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Polypeptide synthesis always begins in the cytosol Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER Polypeptides destined for the ER or for secretion are marked by a signal peptide A signal-recognition particle (SRP) binds to the signal peptide The SRP brings the signal peptide and its ribosome to the ER

LE Ribosomes mRNA Signal peptide Signal- recognition particle (SRP) SRP receptor protein CYTOSOL ER LUMEN Translocation complex Signal peptide removed ER membrane Protein

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Comparing gene expression in prokaryotes and eukaryotes reveals key differences Prokaryotic cells lack a nuclear envelope, allowing translation to begin while transcription progresses In a eukaryotic cell: – The RNA polymerase is different and requires transcription factors – The nuclear envelope separates transcription from translation – Extensive RNA processing occurs in the nucleus

LE RNA polymerase DNA Polyribosome RNA polymerase Direction of transcription mRNA 0.25  m DNA Polyribosome Polypeptide (amino end) Ribosome mRNA (5 end)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Point mutations can affect protein structure and function Mutations are changes in the genetic material Point mutations are chemical changes in just one base pair of a gene The change of a single nucleotide in a DNA template strand leads to production of an abnormal protein

LE Wild-type hemoglobin DNA mRNA Mutant hemoglobin DNA mRNA Normal hemoglobinSickle-cell hemoglobin

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Types of Point Mutations Point mutations within a gene can be divided into two general categories – Base-pair substitutions – Base-pair insertions or deletions

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Substitutions A base-pair substitution replaces one nucleotide and its partner with another pair of nucleotides Missense mutations still code for an amino acid, but not necessarily the right amino acid Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein Missense mutations are more common

LE Base-pair substitution No effect on amino acid sequence U instead of C Missense A instead of G Nonsense U instead of A Stop Amino end Protein 53 Carboxyl end Stop mRNA Wild type

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Insertions and Deletions Insertions and deletions are additions or losses of nucleotide pairs in a gene These mutations affect protein structure more often than substitutions do Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation

LE Base-pair insertion or deletion Frameshift causing immediate nonsense Extra U Missing Frameshift causing extensive missense Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid Missing Stop Amino end Carboxyl end Stop Wild type mRNA Protein 53

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Mutagens Spontaneous mutations can occur during DNA replication, recombination, or repair Spontaneous mutation is 1 in per cell cycle Mutagens are physical or chemical agents that can cause mutations Examples are x-rays, UV light and ethidium bromide

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings What is a gene? revisiting the question A gene occupies a position on a chromosome called a locus A gene is a region of DNA whose final product is either a polypeptide or an RNA molecule Regulatory regions of genes are not transcribed and introns do not code for amino acids

LE TRANSCRIPTION RNA PROCESSING RNA transcript 5 Exon NUCLEUS FORMATION OF INITIATION COMPLEX CYTOPLASM 3 DNA RNA polymerase RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Amino acid tRNA AMINO ACID ACTIVATION 3 mRNA A P E Ribosomal subunits 5 Growing polypeptide E A Activated amino acid Anticodon TRANSLATION Codon Ribosome

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Next Lecture: Bacterial and Viral Genetics