The Molecular Genetics of Gene Expression 9 The Molecular Genetics of Gene Expression

Gene Expression Principles Gene expression involves processes of transcription and translation which result in the production of proteins whose structure is determined by genes The primary structure of proteins is a linear sequence of amino acids held together by peptide bonds

Gene Expression Principles Peptide bonds link the carboxyl group of one amino acid to the amino group of the next amino acid There are twenty naturally occurring amino acids, the fundamental building blocks of proteins The linear sequence of amino acids in proteins is specified by the coding information in specific genes

Gene Expression Principles Polypeptide chains are linear polymers of amino acids There are 20 amino acids each with a unique side chain = R group Colinearity: the linear order of amino acids is encoded in a DNA base sequence The base sequence in DNA specifies the base sequence in RNA transcript

Transcription Transcription = production of messenger RNA (mRNA) complementary to the base sequence of specific genes mRNA differs from DNA in that it is single stranded, contains ribose sugar instead of deoxyribose and the pyrimidine uracil in place of thymine

RNA Synthesis The nucleotide sequence in the transcribed mRNA is complementary to the base sequence in DNA RNA is copied from the template strand which is 3’-to-5’ in the 5’-to-3’ direction = antiparallela RNA synthesis does not require a primer and proceeds by the addition of nucleotides to form mRNA chain

RNA Synthesis Promoter = nucleotide sequence 5’ to the transcription start site which is the initial binding site of RNA polymerase and transcription initiation factors Promoter recognition by RNA polymerase is a prerequisite for transcription initiation

RNA Synthesis Many promoters contain a similar DNA sequence = TATAAT = “TATA” box (-10) is a consensus sequence of many promoters Consensus promoter sequence at -35 = TTGACA Transcription termination sites are inverted repeat sequences which can form loops in RNA = stop signal

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 = “cap”

Eukaryotic Transcription In many eukaryotic genes the coding regions which specify the structure of proteins are interrupted by noncoding segments = “split genes” Coding regions = exons Noncoding regions = introns Primary transcript contains exons and introns; introns are subsequently removed = “splicing”

Eukaryotic Transcription Additional processing involves the addition of a series of adenines at the 3’ end of the transcript = “poly A tail” The processed transcript contains a 5’ cap (7-methylguanosine), adjacent exons and a poly A tail

Eukaryotic Transcription: Splicing RNA splicing occurs in small nuclear ribonucleoprotein particles (snRNPS) in spliceosomes Consensus sequences are located at the 5’ end = donor site and 3’end = acceptor site of the intron “A” nucleotide from branch site in intron attacks “G” at the 5’ terminus cutting the RNA which forms a loop

RNA Transcription: Splicing Next, the 5’ exon moves to the 3’ splice acceptor site where a second cut is made by the spliceosome Exon termini are joined and sealed The loop is released as a lariat structure which is degraded The spliced mRNA contains fused exons with coding information only

RNA Transcription: Splicing Spliceosomes contain protein and specialized small RNAs complementary to the splice junctions to provide specificity to splicing reaction Small nuclear RNAs U1, U2 and U5 recognize splice donor and acceptor sites by complementary base pairing so that intron excision is precise

RNA Splicing Electron micrographs of a DNA-RNA hybrid formed from single-strand template DNA and mRNA show loops of single-strand DNA corresponding to noncoding intron regions spliced from mRNA and poly A tail which is added posttranscriptionally

Translation Translation = genetic information encoded in mRNA specifies the linear sequence of amino acids in the corresponding protein Translation requires mRNA, ribosomes, transfer RNA (tRNA), aminoacyl tRNA synthetases, and initiation, elongation and termination factors

Translation mRNA encodes the information which specifies the primary structure of protein Ribosomes are sites of protein synthesis which contain ribosomal RNA (rRNA) and protein and are organized in two subunits: - small subunit = 30S or 40S (density) - large subunit = 50S or 60S (density)

Translation Transfer RNA (tRNA) = adapter molecule which aligns amino acids in a sequence specified by mRNA Aminoacyl tRNA synthetases = enzymes which attach amino acids to tRNAs to form charged tRNAs Initiation, elongation and termination factors = specialized roles in translation

Translation Initiation complex = mRNA + small ribosomal subunit + tRNA-met attaches to large subunit tRNA-met occupies P (peptidyl) site A second charged tRNA occupies the A (aminoacyl) site Elongation = met is transferred from its tRNA to amino acid at A site Peptide bond links amino acids

Translation Once peptide bond is formed the ribosome shifts one codon along the mRNA to the next codon = translocation, requires EF-G Elongation cycles require EF-Tu-GTP which uses energy to exchange tRNAs on ribosome Peptidyl transferase catalyzes peptide bond formation

Translation tRNAs are covalently attached to specific amino acids by aminoacyl- synthetases and contain anti-codon complementary to the mRNA codon Base pairing between the tRNA anti-codon and the mRNA codon on the ribosome places amino acids in the correct linear sequence in translation

Translation Direction of Synthesis: Template strand of DNA = 3’-to-5’ mRNA = 5’-to-3’ polypeptide = amino terminus (NH2) to carboxy terminus (COOH) Translation termination: No tRNA can bind to stop codon which causes release of polypeptide

Translation Several ribosomes can move in tandem along a messenger RNA to form translation unit=polysome In prokaryotes a single mRNA may contain multiple translation initiation sites = polycistronic mRNA Polycistronic mRNAs allow coordinate regulation of synthesis of more than one protein

Translation: Genetic Code Translation involves the synthesis of proteins consisting of a chain of amino acids whose sequence is specified by the coding information in mRNA mRNA carries the “genetic code” = chemical information originating in DNA which specifies the primary structure of proteins

Translation: Genetic Code Triplet code = three bases in RNA code for a single amino acid = codon There are 64 triplet codons which can be formed from 4 bases: - 61 codons specify 20 amino acids; genetic code is redundant - 3 are chain terminating “stop” codons which end translation

Genetic Code “AUG” is the initiator codon which specifies the placement of methionine as the first amino acid 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

Genetic Code Genetic evidence for a triplet code comes from three-base insertions and deletions Genetic code specifies a reading frame in mRNA in which bases are accessed in linear triplet units Frameshift mutations: alter reading frame by adding or deleting non-multiple of three bases