DNA Transcription and Translation A factory for proteins.

Comparing RNA and DNA DNA can replicate itself precisely and contain information in the specific sequence of its bases. RNA and DNA are very similar molecules. 5-carbon sugar in RNA is Ribose RNA contains Uracil instead of Thymine DNA is double stranded and RNA is single stranded with folded complex secondary and tertiary structures. DNA molecules are always longer than RNA molecules. DNA is more stable than RNA. There are several classes of RNA.

Messenger RNA mRNA carries the message. The linear amino acid sequence (primary) is encoded in the DNA. But the DNA does not make the proteins directly. mRNA is the link between gene and protein. The information contained in the mRNA is written in the genetic code. The genetic code is UNIVERSAL. The beginning of the mRNA is always on the 5’ end and that is where the synthesis of the proteins starts. It takes three nucleotides to code for one amino acid. The mRNA has a 5’ leader, a coding region, introns, and a 3’ trailer. After transcription the mRNA is modified to go inside the cytoplasm. A 5’ cap is added as well as a 3’ poly A tail. Introns are spliced out. Only expressed regions (exons) are kept.

Basic Principles of Transcription and Translation RNA is the intermediate between genes and the proteins for which they code 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 © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

EXPERIMENT RESULTS CONCLUSION Fig. 17-2 Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Minimal medium RESULTS Classes of Neurospora crassa Wild type Class I mutants Class II mutants Class III mutants Minimal medium (MM) (control) MM + ornithine Condition MM + citrulline MM + arginine (control) Figure 17.2 Do individual genes specify the enzymes that function in a biochemical pathway? CONCLUSION Class I mutants (mutation in gene A) Class II mutants (mutation in gene B) Class III mutants (mutation in gene C) Wild type Precursor Precursor Precursor Precursor Gene A Enzyme A Enzyme A Enzyme A Enzyme A Ornithine Ornithine Ornithine Ornithine Gene B Enzyme B Enzyme B Enzyme B Enzyme B Citrulline Citrulline Citrulline Citrulline Gene C Enzyme C Enzyme C Enzyme C Enzyme C Arginine Arginine Arginine Arginine

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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig. 17-3 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

The Genetic Code The genetic code is the same for all organisms (UNIVERSAL) A codon is a “word” in DNA/RNA language. It is formed by three nucleotides. There are a lot of synonymous codons. The genetic code is “redundant” AUG is always the start codon for translation and there are three stop codons to end translation.

Gene 2 Gene 1 Gene 3 DNA template strand mRNA Codon TRANSLATION Fig. 17-4 Gene 2 DNA molecule Gene 1 Gene 3 DNA template strand TRANSCRIPTION Figure 17.4 The triplet code mRNA Codon TRANSLATION Protein Amino acid

(a) Tobacco plant expressing a firefly gene (b) Pig expressing a Fig. 17-6 Figure 17.6 Expression of genes from different species (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene

Key Words and definitions Transcription describes the synthesis of RNA on a DNA template Translation is the synthesis of protein on a mRNA template Coding region of part of the gene that represents a protein sequence. Codon is a triplet of bases that represents an amino acid or a termination signal The antisense strand (template) is complementary to the sense strand and is used as a template to synthesize RNA The coding strand (sense) has the same sequence as the mRNA and is related to the protein synthesized.

RNA Polymerase Binding and Initiation of Transcription Promoters signal the initiation of RNA synthesis Transcription factors mediate the binding of RNA polymerase and the 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Promoters, terminators and start point

Several transcription factors must bind to the DNA before RNA Fig. 17-8 1 A eukaryotic promoter includes a TATA box Promoter Template 5 3 3 5 TATA box Start point Template DNA strand 2 Several transcription factors must bind to the DNA before RNA polymerase II can do so. Transcription factors 5 3 3 5 3 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. Figure 17.8 The initiation of transcription at a eukaryotic promoter RNA polymerase II Transcription factors 5 3 3 5 5 RNA transcript Transcription initiation complex

Only one strand of DNA is transcribed

Sense and antisense strands

Completed RNA transcript Fig. 17-7 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase 1 Initiation Elongation Nontemplate strand of DNA RNA nucleotides 5 3 RNA polymerase 3 5 RNA transcript Template strand of DNA Unwound DNA 3 2 Elongation 3 end Rewound DNA 5 5 3 3 3 5 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 Direction of transcription (“downstream”) RNA transcript Template strand of DNA 3 Termination Newly made RNA 5 3 3 5 5 3 Completed RNA transcript

Protein-coding segment Polyadenylation signal 5 3 Fig. 17-9 Protein-coding segment Polyadenylation signal 5 3 G P P P AAUAAA AAA … AAA 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail Figure 17.9 RNA processing: addition of the 5 cap and poly-A tail

Split Genes and 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

exons spliced together Coding segment Fig. 17-10 5 Exon Intron Exon Intron Exon 3 Pre-mRNA 5 Cap Poly-A tail 1 30 31 104 105 146 Introns cut out and exons spliced together Coding segment mRNA 5 Cap Poly-A tail 1 146 Figure 17.10 RNA processing: RNA splicing 5 UTR 3 UTR

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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Fig. 17-11-1 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Figure 17.11 The roles of snRNPs and spliceosomes in pre-mRNA splicing

RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Fig. 17-11-2 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Figure 17.11 The roles of snRNPs and spliceosomes in pre-mRNA splicing

RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Fig. 17-11-3 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Figure 17.11 The roles of snRNPs and spliceosomes in pre-mRNA splicing Spliceosome components Cut-out intron mRNA 5 Exon 1 Exon 2

Ribozymes Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Three properties of RNA enable it to function as an enzyme It can form a three-dimensional structure because of its ability to base pair with itself Some bases in RNA contain functional groups RNA may hydrogen-bond with other nucleic acid molecules Copyright © 2008 Pearson Education Inc., publishing as Pearson 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 © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Exon shuffling may result in the evolution of new proteins 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 Exon shuffling may result in the evolution of new proteins Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription Fig. 17-12 Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Figure 17.12 Correspondence between exons and protein domains Domain 2 Domain 1 Polypeptide

There are three types of RNA (and many others)

Transfer RNA It is the translator of the code. Short length RNA (90 nucleotides) Introns are spliced out and chemically transformed bases are added and replace others. The tRNA is coiled and loops back on itself in a defined structure. It is linked covalently to an amino acid. The tRNA possesses an anti-codon.

tRNA

Aminoacyl-tRNA The aminoacyl-tRNA synthase adds the correct amino acid to the corresponding tRNA.

Aminoacyl-tRNA Amino acid synthetase (enzyme) tRNA Aminoacyl-tRNA Fig. 17-15-4 Aminoacyl-tRNA synthetase (enzyme) Amino acid P P P Adenosine ATP P Adenosine tRNA P P i Aminoacyl-tRNA synthetase P i P i tRNA Figure 17.15 An aminoacyl-tRNA synthetase joining a specific amino acid to a tRNA P Adenosine AMP Computer model Aminoacyl-tRNA (“charged tRNA”)

Ribosomal RNA Ribosomal RNA: contributes to the structure of Ribosomes. In eukaryotes rRNA is transcribed exclusively in the nucleolus. The primary transcript is processed by an enzyme to produce the shorter rRNA constituting the ribosome. Ribosomes are made of 40S subunit (small) and 60S subunit (large). Proteins make up about half of the ribosome. The complete ribosome is 80S.

Ribosomes

In Prokaryotes

(a) Computer model of functioning ribosome Fig. 17-16a Growing polypeptide Exit tunnel tRNA molecules Large subunit E P A Small subunit Figure 17.16 The anatomy of a functioning ribosome 5 3 mRNA (a) Computer model of functioning ribosome

(b) Schematic model showing binding sites Fig. 17-16b P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) E P A Large subunit mRNA binding site Small subunit (b) Schematic model showing binding sites Growing polypeptide Amino end Next amino acid to be added to polypeptide chain Figure 17.16 The anatomy of a functioning ribosome E tRNA mRNA 3 Codons 5 (c) Schematic model with mRNA and tRNA

A ribosome has three binding sites for tRNA: The P site holds the tRNA that carries the growing polypeptide chain The A site holds the tRNA that carries the next amino acid to be added to the chain The E site is the exit site, where discharged tRNAs leave the ribosome Copyright © 2008 Pearson Education Inc., publishing as Pearson 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 that completes the translation initiation complex Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Translation initiation complex Fig. 17-17 Large ribosomal subunit 3 U C 5 A P site Met 5 A Met U G 3 Initiator tRNA GTP GDP E A mRNA 5 5 3 3 Start codon Figure 17.17 The initiation of translation Small ribosomal subunit mRNA binding site Translation initiation complex

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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Amino end of polypeptide E 3 mRNA 5 Fig. 17-18-1 P A site site Figure 17.18 The elongation cycle of translation

GDP Amino end of polypeptide E 3 mRNA 5 E P A Fig. 17-18-2 P A site GTP GDP E P A Figure 17.18 The elongation cycle of translation

GDP Amino end of polypeptide E 3 mRNA 5 E P A E P A Fig. 17-18-3 P A site A site 5 GTP GDP E P A Figure 17.18 The elongation cycle of translation E P A

GDP GDP Amino end of polypeptide E 3 mRNA Ribosome ready for Fig. 17-18-4 Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P site A site 5 GTP GDP E E P A P A Figure 17.18 The elongation cycle of translation GDP GTP E P A

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 reaction releases the polypeptide, and the translation assembly then comes apart Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Release factor 3 5 Stop codon (UAG, UAA, or UGA) Fig. 17-19-1 Figure 17.19 The termination of translation

Release factor Free polypeptide 3 3 2 5 5 Stop codon Fig. 17-19-2 Release factor Free polypeptide 3 3 2 5 5 GTP Stop codon (UAG, UAA, or UGA) 2 GDP Figure 17.19 The termination of translation

Release factor Free polypeptide 5 3 3 3 2 5 5 Stop codon Fig. 17-19-3 Release factor Free polypeptide 5 3 3 3 2 5 5 GTP Stop codon (UAG, UAA, or UGA) 2 GDP Figure 17.19 The termination of translation

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 © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Protein Folding and Post-Translational Modifications During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape Proteins may also require post-translational modifications before doing their job Some polypeptides are activated by enzymes that cleave them Other polypeptides come together to form the subunits of a protein Copyright © 2008 Pearson Education Inc., publishing as Pearson 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 Ribosomes are identical and can switch from free to bound Copyright © 2008 Pearson Education Inc., publishing as Pearson 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 Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Ribosome mRNA Signal peptide ER membrane Signal peptide removed Fig. 17-21 Ribosome mRNA Signal peptide ER membrane Signal peptide removed Signal- recognition particle (SRP) Protein CYTOSOL Translocation complex Figure 17.21 The signal mechanism for targeting proteins to the ER ER LUMEN SRP receptor protein

Point mutations 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 base pair of a gene The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Wild-type hemoglobin DNA Mutant hemoglobin DNA 3 C T T 5 3 C A T 5 Fig. 17-22 Wild-type hemoglobin DNA Mutant hemoglobin DNA 3 C T T 5 3 C A T 5 5 G A A 3 5 G T A 3 mRNA mRNA 5 G A A 3 5 G U A 3 Figure 17.22 The molecular basis of sickle-cell disease: a point mutation Normal hemoglobin Sickle-cell hemoglobin Glu Val

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 © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Silent (no effect on amino acid sequence) Fig. 17-23a Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of G 3 5 5 3 Figure 17.23 Types of point mutations U instead of C 5 3 Stop Silent (no effect on amino acid sequence)

Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Fig. 17-23b Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end T instead of C 3 5 5 3 Figure 17.23 Types of point mutations A instead of G 5 3 Stop Missense

Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Fig. 17-23c Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of T 3 5 5 3 Figure 17.23 Types of point mutations U instead of A 5 3 Stop Nonsense

Frameshift causing immediate nonsense (1 base-pair insertion) Fig. 17-23d Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end Extra A 3 5 5 3 Figure 17.23 Types of point mutations Extra U 5 3 Stop Frameshift causing immediate nonsense (1 base-pair insertion)

Frameshift causing extensive missense (1 base-pair deletion) Fig. 17-23e Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 3 5 5 3 Figure 17.23 Types of point mutations missing 5 3 Frameshift causing extensive missense (1 base-pair deletion)

Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Fig. 17-23f Wild type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end missing 3 5 5 3 Figure 17.23 Types of point mutations missing 5 3 Stop No frameshift, but one amino acid missing (3 base-pair deletion)