Gene Expression: From Gene to Protein 14 Gene Expression: From Gene to Protein

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

Basic Principles of Transcription and Translation RNA is the bridge between DNA and protein synthesis RNA is chemically similar to DNA, but RNA has a ribose sugar and the base uracil (U) rather than thymine (T) RNA is usually single-stranded Getting from DNA to protein requires two stages: transcription and translation 3 3

Transcription is the synthesis of RNA using information in DNA Transcription produces messenger RNA (mRNA) Translation is the synthesis of a polypeptide, using information in the mRNA Ribosomes are the sites of translation 4 4

In prokaryotes, translation of mRNA can begin before transcription has finished In eukaryotes, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield the finished mRNA Eukaryotic mRNA must be transported out of the nucleus to be translated 5 5

Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA DNA Figure 14.4 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Ribosome Figure 14.4 Overview: the roles of transcription and translation in the flow of genetic information TRANSLATION Polypeptide Polypeptide (a) Bacterial cell (b) Eukaryotic cell 6

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 How many nucleotides correspond to an amino acid? 7 7

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 mRNA These words are then translated into a chain of amino acids, forming a polypeptide 8 8

DNA template strand 3 5 A C C A A A C C G A G T T G G T T T G G C T Figure 14.5 DNA template strand 3 5 A C C A A A C C G A G T T G G T T T G G C T C A 5 3 TRANSCRIPTION U G G U U U G G C U C A mRNA 5 3 Codon Figure 14.5 The triplet code TRANSLATION Protein Trp Phe Gly Ser Amino acid 9

The template strand is always the same strand for any given gene During transcription, one of the two DNA strands, called the template strand, provides a template for ordering the sequence of complementary nucleotides in an RNA transcript The template strand is always the same strand for any given gene 10 10

During translation, the mRNA base triplets, called codons, are read in the 5 to 3 direction Each codon specifies the amino acid (one of 20) to be placed at the corresponding position along a polypeptide 11 11

Cracking the Code All 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 12 12

Codons are read one at a time in a nonoverlapping fashion 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 13 13

First mRNA base (5 end of codon) Third mRNA base (3 end of codon) Figure 14.6 Second mRNA base U C A G UUU UCU UAU UGU U Phe Tyr Cys UUC UCC UAC UGC C U Ser UUA UCA UAA Stop UGA Stop A Leu UUG UCG UAG Stop UGG Trp G CUU CCU CAU CGU U His CUC CCC CAC CGC C C Leu Pro Arg CUA CCA CAA CGA A Gln CUG CCG CAG CGG G First mRNA base (5 end of codon) Third mRNA base (3 end of codon) AUU ACU AAU AGU U Asn Ser AUC IIe ACC AAC AGC C A Thr Figure 14.6 The codon table for mRNA AUA ACA AAA AGA A Lys Arg AUG Met or start ACG AAG AGG G GUU GCU GAU GGU U Asp GUC GCC GAC GGC C G Val Ala Gly GUA GCA GAA GGA A Glu GUG GCG GAG GGG G 14

Evolution of the Genetic Code The genetic code is nearly universal, shared by the simplest bacteria and the most complex animals Genes can be transcribed and translated after being transplanted from one species to another 15 15

Concept 14.2: Transcription is the DNA-directed synthesis of RNA: a closer look Transcription is the first stage of gene expression 16 16

Molecular Components of Transcription RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and joins together the RNA nucleotides RNA polymerases assemble polynucleotides in the 5 to 3 direction However, RNA polymerases can start a chain without a primer Animation: Transcription Introduction 17 17

Completed RNA transcript Figure 14.8-3 Promoter Transcription unit 5 3 3 5 Start point RNA polymerase 1 Initiation 5 3 3 5 Unwound DNA RNA transcript Template strand of DNA 2 Elongation Rewound DNA 5 3 3 3 5 5 Figure 14.8-3 The stages of transcription: initiation, elongation, and termination (step 3) Direction of transcription (“downstream”) RNA transcript 3 Termination 5 3 3 5 5 3 Completed RNA transcript 18

The stretch of DNA that is transcribed is called a transcription unit The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator The stretch of DNA that is transcribed is called a transcription unit 19 19

Synthesis of an RNA Transcript The three stages of transcription Initiation Elongation Termination 20 20

RNA 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 RNA polymerase and the initiation of transcription 21 21

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 22 22

DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Figure 14.UN02 DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 14.UN02 In-text figure, transcription, p. 275 Ribosome TRANSLATION Polypeptide 23

Several transcription factors bind to DNA. 3 5 Figure 14.9 Promoter Nontemplate strand DNA 5 T A T A A A A 3 3 A T A T T T T 5 1 A eukaryotic promoter TATA box Start point Template strand Transcription factors 5 3 2 Several transcription factors bind to DNA. 3 5 RNA polymerase II Transcription factors 3 Transcription initiation complex forms. Figure 14.9 The initiation of transcription at a eukaryotic promoter 5 3 3 3 5 5 RNA transcript Transcription initiation complex 24

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 40 nucleotides per second in eukaryotes A gene can be transcribed simultaneously by several RNA polymerases 25 25

Direction of transcription Template strand of DNA Figure 14.10 Nontemplate strand of DNA RNA nucleotides RNA polymerase T C C A A A 3 T 5 U C T 3 end T G U A G A C C A U C C A C A 5 A 3 T A G G T T Figure 14.10 Transcription elongation 5 Direction of transcription Template strand of DNA Newly made RNA 26

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 mRNA can be translated without further modification In eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence; the RNA transcript is released 10–35 nucleotides past this polyadenylation sequence 27 27

Concept 14.3: Eukaryotic cells modify RNA after transcription Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm During RNA processing, both ends of the primary transcript are altered Also, usually some interior parts of the molecule are cut out and the other parts spliced together 28 28

Alteration of mRNA Ends Each end of a pre-mRNA molecule is modified in a particular way The 5 end receives a modified G nucleotide 5 cap The 3 end gets a poly-A tail These modifications share several functions Facilitating the export of mRNA to the cytoplasm Protecting mRNA from hydrolytic enzymes Helping ribosomes attach to the 5 end 29 29

DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Figure 14.UN03 DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 14.UN03 In-text figure, RNA processing, p. 277 Ribosome TRANSLATION Polypeptide 30

Polyadenylation signal Figure 14.11 50–250 adenine nucleotides added to the 3 end A modified guanine nucleotide added to the 5 end Polyadenylation signal Protein-coding segment 5 3 G P P P AAUAAA AAA … AAA Start codon Stop codon 5 Cap 5 UTR 3 UTR Poly-A tail Figure 14.11 RNA processing: addition of the 5' cap and poly-A tail 31

Split Genes and RNA Splicing Most eukaryotic mRNAs 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 RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence 32 32

exons spliced together Figure 14.12 Pre-mRNA Intron Intron 5 Cap Poly-A tail 1–30 31–104 105– 146 Introns cut out and exons spliced together mRNA 5 Cap Poly-A tail 1–146 Figure 14.12 RNA processing: RNA splicing 5 UTR 3 UTR Coding segment AAUAAA 33

This process is called alternative RNA splicing Many genes can give rise to two or more different polypeptides, depending on which segments are used as exons This process is called alternative RNA splicing RNA splicing is carried out by spliceosomes Spliceosomes consist of proteins and small RNAs 34 34

Spliceosome Small RNAs 5 Pre-mRNA Exon 1 Exon 2 Intron Spliceosome Figure 14.13 Spliceosome Small RNAs 5 Pre-mRNA Exon 1 Exon 2 Intron Figure 14.13 A spliceosome splicing a pre-mRNA Spliceosome components mRNA Cut-out intron 5 Exon 1 Exon 2 35

Ribozymes Ribozymes are RNA molecules that function as enzymes RNA splicing can occur without proteins, or even additional RNA molecules The introns can catalyze their own splicing 36 36