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Agenda Brief Review of Transcription and Translation Self Assessment Questions Question 2

8 The Molecular Genetics of Gene Expression

Four Basic Molecular Genetic Process: (1) Transcription, (2) RNA processing, (3) mRNA translation, (4) DNA replication WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

5 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

6 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

7 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

8 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

Important points: One strand of DNA acts as the template - determining the order in which ribonucleoside triphosphate monomers (rNTPs) are polymerized to form a complementary RNA chain. Polymerization reaction is catalyzed by RNA polymerase. A phosphodiester bond is formed between the 3’ oxygen and the alpha phosphate of the incoming rNTP. RNA molecules are always synthesized in a 5’ to 3’ direction. TRANSCRIPTION: Polymerization of ribonucleotides by RNA polymerase during trancription WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

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 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

TRANSCRIPTION: WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

TRANSCRIPTION: WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

Gene Organization in Prokaryotes and Eukaryotes In prokaryotes, genes devoted to a single metabolic goal are most often found in a contiguous array in the DNA called an operon. In eukaryotes, these genes are most often separated. WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

17 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

mRNA Processing in Eukaryotes: Important points: Exons - coding sequences Introns- non-coding sequences 5’ end is processed by addition of a 5’methylated cap 3’ end is processed by cleavage by an endonuclease to yield a 3’ hydroxyl group where adenylic acid residues are added by poly (A) polymerase to give a poly(A) tail ( As) RNA splicing - introns excised and exonic sequences ligated together WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

mRNA Processing in Eukaryotes: 5’ methylated cap Important points: Cap protects an mRNA from enzymatic degradation and assists in export to cytoplasm Cap is bound by a protein factor required to begin translation in the cytoplasm. WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

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 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

Alternative RNA Splicing Increases the Number of Proteins Expressed from a Single Eukaryotic Gene Alternative Splicing - permits the expression of multiple related proteins from a single gene Isoforms - different forms of a protein produced by alternative splicing Nearly 60% of human genes are alternatively spliced WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

Four Basic Molecular Genetic Process: (1) Transcription, (2) RNA processing, (3) mRNA translation, (4) DNA replication WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

24 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

25 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

26 Fig. 8.3

27 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

28 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.

29 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

The genetic code can be read in three different frames. WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

31 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

The Three Roles of RNA in Protein Synthesis 1.) Messenger RNA (mRNA) -transcribed from DNA -three-nucleotide sequences form codons, specifying an amino acid 2.) Transfer RNA (tRNA) - deciphers the codons in mRNA -each amino acid has its own subset of tRNAs -each tRNA contains a 3- nucleotide sequence, an anticodon, that can base-pair with its complementary codon in the mRNA. 3.) Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes. -physically move along an mRNA molecule, catalyze the assembly of amino acids into polypeptide chains. -Ribosomes are composed of a large and a small subunit, each of which contains its own rRNA molecule (s). WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

Translation: Two-step decoding process for translating nucleic acid sequences in mRNA into amino acid sequences in proteins. 1.) An aminoacyl-tRNA synthetase first couples a specific amino acid, via a high- energy bond to the hydroxyl of the corresponding tRNA. 2.) A three-base sequence in the tRNA (the anticodon) then base-pairs with a codon in the mRNA specifying the attached amino acid. WH Freeman, Molecular Cell Biology, 5 th Edition, 2004.

34 Fig. 8.22

35 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

36 Fig. 8.24

37 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

Questions? 38