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8 The Molecular Genetics of Gene Expression Gene Expression Steps • Gene expression is the process by which information contained in genes is decoded to produce other molecules that determine the phenotypic traits of organisms • The principal steps in gene expression are: Transcription: RNA molecules are synthesized by an enzyme, RNA polymerase, which uses a segment of a single strand of DNA as a template strand to produce a strand of RNA complementary in base sequence to the template DNA 2 Gene Expression Steps • In the nucleus of eukaryotic cells, the RNA usually undergoes chemical modification called RNA processing • Translation: the processed RNA molecule is used to specify the order in which amino acids are joined together to form a polypeptide chain. In this manner, the amino acid sequence in a polypeptide is a direct consequence of the base sequence in the DNA • The protein made is called the gene product 3 Polypeptides • Polypeptide chains are linear polymers of amino acids • There are twenty naturally occurring amino acids, the fundamental building blocks of proteins • Peptide bonds link the carboxyl group of one amino acid to the amino group of the next amino acid • The sequence of amino acids in proteins is specified by the coding information in specific genes 4 Fig. 8.3 5 Protein Domains • Most polypeptides include regions that can fold in upon themselves to acquire well-defined structures = domains • Domains interact with each other • The domains often have specialized functions • The individual domains in a protein usually have independent evolutionary origins, they come together in various combinations to create genes with novel functions via duplication of their coding regions and genomic rearrangements 6 Protein Domains • Domains can be identified through computer analysis of the amino acid sequence • Vertebrate genomes have relatively few proteins or protein domains not found in other organisms. Their complexity arises in part from innovations in bringing together preexisting domains to create novel proteins that have more complex domain architectures than those found in other organisms. 7 Colinearity • The linear order of nucleotides in a gene determines the linear order of amino acids in a polypeptide • This attribute of genes and polypeptides is called colinearity, which means that the sequence of base pairs in DNA determines the sequence of amino acids in the polypeptide in a colinear, or point-to-point, manner • Colinearity is universally found in prokaryotes • In eukaryotes, noninformational DNA sequences interrupt the continuity of most genes 8 Transcription • Transcription = the process of synthesis of an RNA molecule copied from the segment of DNA that constitutes the gene • RNA differs from DNA in that it is single stranded, contains ribose sugar instead of deoxyribose and the pyrimidine uracil in place of thymine Fig. 8.5 9 RNA Synthesis • The nucleotide sequence in the transcribed mRNA is complementary to the base sequence in DNA • In the synthesis of RNA, a sugar–phosphate bond is formed between the 3’- hydroxyl group of one nucleotide and the 5’- OH triphosphate of the next nucleotide in line • RNA synthesis does not require a primer • The enzyme used in transcription is RNA polymerase 10 Fig. 8.6a,b 11 Fig. 8.6c 12 RNA Polymerases • RNA polymerases are large, multisubunit complexes whose active form is called the RNA polymerase holoenzyme • Bacterial cells have only one RNA polymerase holoenzyme, which contains six polypeptide chains • Eukaryotes have several types of RNA polymerase • RNA polymerase I transcribes ribosomal RNA. • RNA polymerase II - all protein-coding genes as well as the genes for small nuclear RNAs • RNA polymerase III - tRNA genes and the 5S component of rRNA 13 RNA Synthesis • Particular nucleotide sequences define the beginning and end of a gene • Promoter = nucleotide sequence 20-200 bp long—is the initial binding site of RNA polymerase and transcription initiation factors • Promoter recognition by RNA polymerase is a prerequisite for transcription initiation Fig. 8.8 14 RNA Synthesis • The consensus promoter regions in E. coli are TTGACA (-35), centered approximately 35 base pairs upstream from the transcription start site (numbered the +1 site) TATAAT (-10) = “TATA” box: 10 base pairs upstream Transcription termination sites are inverted repeat sequences which can form loops in RNA = stop signal 15 Fig. 8.9 16 Eukaryotic Transcription • Eukaryotic transcription involves the synthesis of RNA specified by DNA template strand to form a primary transcript • Primary transcript is processed to form mRNA which is transported to the cytoplasm • The first processing step adds 7- methylguanosine to 5’-end of the primary transcript = cap 17 Fig. 8.12 18 Eukaryotic Transcription • Translation of an mRNA molecule rarely starts exactly at one end and proceeds to the other end: initiation of protein synthesis may begin many nucleotides downstream from the 5’-end • The 5’ untranslated region followed by an open reading frame (ORF), which specifies polypeptide chain • In many eukaryotic genes ORFs are interrupted by noncoding segments—introns 19 Eukaryotic Transcription • Coding regions interrupted by introns—exons • Primary transcript contains exons and introns; introns are subsequently removed by splicing • The 3’-end of an mRNA molecule following the ORF also is not translated; it is called the 3’ untranslated region • The 3’- end is usually modified by the addition of a sequence called the poly-A tail 20 Splicing • RNA splicing occurs in nuclear particles known as spliceosomes • The specificity of splicing comes from the five small snRNP—RNAs denoted U1, U2, U4, U5, and U6, which contain sequences complementary to the splice junctions 21 Fig. 8.13 22 Splicing Human genes tend to be very long even though they encode proteins of modest size The average human gene occupies 27 kb of genomic DNA, yet only 1.3 kb (~ 5 %) is used to encode amino acids The correlation between exons and domains found in some genes suggests that the genes were originally assembled from smaller pieces The model of protein evolution through the combination of different exons is called the exon shuffle model 23 Translation • The synthesis of every protein molecule in a cell is directed by an mRNA originally copied from DNA • Protein production includes two kinds of processes: • information-transfer processes, in which the RNA base sequence determines an amino acid sequence • chemical processes, in which the amino acids are linked together. • The complete series of events is called translation 24 Translation • The translation system consists of five major components: Messenger RNA: mRNA is needed to provide the coding sequence of bases that determines the amino acid sequence in the resulting polypeptide chain Ribosomes are particles on which protein synthesis takes place Transfer RNA: tRNA is a small adaptor molecule that translates codons into amino acid Aminoacyl-tRNA synthetases: set of molecules catalyzes the attachment of a particular amino acid to its corresponding tRNA molecule Initiation, elongation, and termination factors 25 Translation: Initiation • In eukaryotes, initiation takes place by scanning the mRNA for an initiation codon • In the translation initiation, the 5’ cap on the mRNA is instrumental • The elongation factor eIF4F binds to the cap and recruits eIF4A and eIF4B • This creates a binding site for a charged tRNAMet (an initiator tRNA), bound with elongation factor eIF2, and a small 40S ribosomal subunit together with eIF3 and eIF5 • These components all come together at the 5’ cap and form the 48S initiation complex 26 Fig. 8.15a, b 27 Translation: Initiation • The initiation complex moves along the mRNA in the 3’ direction, scanning for the first of the initial methionine codon AUG • At this point eIF5 causes the release of all the initiation factors and the recruitment of a large 60S ribosomal subunit • This subunit includes three binding sites for tRNA molecules: the E (exit) site, the P (peptidyl) site, and the A (aminoacyl) site. • At the beginning the tRNAMet is located in the P site and the A site is the next in line to be occupied. • The tRNA binding is accomplished by hydrogen bonding between the AUG codon in the mRNA and the three-base anticodon in the tRNA. 28 Fig. 8.15b, c 29 Translation: Elongation • In the first step of elongation, the 40S subunit moves one codon farther along the mRNA, and the charged tRNA corresponding to the new codon is brought into the A site on the 60S subunit • A peptidyl transferase activity catalyzes a coupled reaction in which the bond connecting the methionine to the tRNAMet is transferred to the amino group of the next amino acid, forming the first peptide bond • In the next step, the 60S subunit swings forward to catch up with the 40S, and at the same time the tRNAs in the P and A sites of the large subunit are shifted to the E and P sites, respectively 30 Fig. 8.16a, b 31 Fig. 8.16c, d 32 Translation: Elongation • One cycle of elongation is now completed, and the entire procedure is repeated for the next codon • Eukaryotes synthesize a polypeptide chain at the rate of about 15 amino acids per second • Elongation in prokaryotes is a little faster (about 20 amino acids per second), but the essential processes are very similar 33 Translation: Termination • When a stop codon is encountered, the tRNA holding the polypeptide remains in the P site, and a release factor (RF) binds with the ribosome. • GTP hydrolysis provides the energy to cleave the polypeptide from the tRNA to which it is attached • The 40S and 60S subunits are recycled to initiate translation of another mRNA • Eukaryotes have only one release factor that recognizes all three stop codons: UAA, UAG, and UGA • There are three release factors in E. coli 34 Translation • The mRNA is translated in the 5’-to-3’ direction. The polypeptide is synthesized from the amino end toward the carboxyl end • Most polypeptide chains fold correctly as they exit the ribosome: they pass through a tunnel in the large ribosomal subunit that is long enough to include about 35 amino acids • Emerging from the tunnel, protein enters into a sort of cradle formed by a protein associated with the ribosome: it provides a space where the polypeptide is able to undergo its folding process. • The proper folding of more complex polypeptides is aided by proteins called chaperones and chaperonins 35 Fig. 8.22 36 Translation: Prokaryotes • In prokaryotes, mRNA molecules have no cap, and there is no scanning mechanism • In E. coli, IF-1 and IF-3 initiation factors interact with the 30S subunit and IF-2 binds with a special tRNA charged with formylmethionine tRNAfMet • These components bind with an mRNA at the ribosome-binding site, RBS or the Shine–Dalgarno sequence. Together, they recruit a 50S sub-unit • mRNA molecules contain information for the amino acid sequences of several different proteins; such a molecule is called a polycistronic mRNA 37 Translation: Prokaryotes • Cistron = DNA sequence that encodes a single polypeptide chain • In a polycistronic mRNA, each protein coding region is preceded by its own ribosome-binding site and AUG initiation codon • After the synthesis of one polypeptide is finished, the next along the way is translated • The genes contained in a polycistronic mRNA often encode the different proteins of a metabolic 38 pathway. Genetic Code • The genetic code is the list of all codons and the amino acids that they encode • Main features of the genetic code were proved in genetic experiments carried out by F.Crick and collaborators: • Translation starts from a fixed point • There is a single reading frame maintained throughout the process of translation • Each codon consists of three nucleotides • Code is nonoverlapping • Code is degenerate: each amino acid is specified by more than one codon 39 Fig. 8.24 40 Genetic Code • Most of the codons were determined from in vitropolypeptide synthesis • Genetic code is universal = the same triplet codons specify the same amino acids in all species • Mutations occur when changes in codons alter amino acids in proteins 41