Translation - 2021-1.pdf

Encoding proteins: Translation Dr Mark Carlile Dale 106 [email protected] Dr Mark Carlile : Transcription & Translation 1 Overview of my lectures on PHA111 All of my lectures are broadly focused on molecular biology: Wk 25: Nucleic acid biochemistry Wk 26: DNA replication Wk 26: Transcri...

Encoding proteins: Translation Dr Mark Carlile Dale 106 [email protected] Dr Mark Carlile : Transcription & Translation 1 Overview of my lectures on PHA111 All of my lectures are broadly focused on molecular biology: Wk 25: Nucleic acid biochemistry Wk 26: DNA replication Wk 26: Transcription Wk 27: Translation (protein synthesis) Wk 27: Protein folding and sorting Wk 28: Biotechnology Wk 28: Recap and some biological therapeutics stuff Dr Mark Carlile | Nucleic acid biochemistry 2 The Central Dogma of Molecular Biology Cellular function Lecture 1 Lecture 2 Lecture 3 Look at how the genetic material is controllably replicated prior to cell division Look at the functional genome and its generation (RNA) Look at how the genome is translated into working parts Dr Mark Carlile : Transcription & Translation 3 The Central Dogma of Molecular Biology Transcription is the enzymatic synthesis of RNA from a DNA template and forms the first step in gene expression Generation of a messenger RNA (mRNA) Translation is the enzymatic synthesis of protein from an transcribed gene sequence into a functional RNA molecule mRNA Dr Mark Carlile : Transcription & Translation 4 Protein structure The simplest level of protein structure, primary structure, is simply the sequence of amino acids in a polypeptide chain. Secondary structure, refers to local folded structures that form within a polypeptide due to interactions between atoms of the backbone. The overall three-dimensional structure of a polypeptide is called its tertiary structure. The tertiary structure is primarily due to interactions between the R groups of the amino acids that make up the protein Many proteins are made up of a single polypeptide chain and have only three levels of structure. However, some proteins are made up of multiple polypeptide chains, also known as subunits. When these subunits come together, they give the protein its quaternary structure Dr Mark Carlile : Transcription & Translation 5 Amino acids More on this in wk 16 (Dr Ahmed) Dr Mark Carlile : Transcription & Translation 6 Translation Translation is the process whereby ribosomes read the transcribed mRNA and generate a protein In prokaryotes, translation is cotranscriptional, i.e. they happen at the same time In eukaryotes, translation occurs after transcription (but some elements of the process are co-transcriptional) Translation happens in the cytoplasm Dr Mark Carlile : Transcription & Translation 7 The Ribosome Ribosomes are responsible for reading the mRNA sequence and assembling the protein based upon the message Ribosomes have a diameter of approx 10 nm and are composed of ribosomal RNA (rRNA; 65%) and ribosomal proteins (35%) The different units of the ribosome are characterised based on the rate at which they sediment (via ultracentrifugation) Measured in Svedberg units (S) Dr Mark Carlile : Transcription & Translation 8 The Ribosome Under optimal conditions mixtures of the rRNAs and protein will self-assemble into a ribosome The information for the ribosome structure is inherent in the sub-components The RNAs have both structural and catalytic activity The activity of ribosomes involves a complex interplay between rRNA and ribosomal proteins The small ribosomal subunit contains the decoding centre which is needed for reading the mRNA The large ribosomal subunit contains the peptidyl transferase centre (catlytic RNA) When multiple ribosomes are loaded onto a mRNA strand they are known as a polyribosome Dr Mark Carlile : Transcription & Translation 9 The Ribosome Transfer RNAs occupy the A- P- and E-sites: Transfer RNAs (tRNAs) carry amino acids A-site: acceptor site, where the amino-acyl tRNA lands P-site: peptidyl-tRNA site, occupied by the last amino-acid added E-site: exit site, where the tRNA sits once it has transferred its amino-acid – the tRNA leaves the ribosome from the E-site Although the bulk of each of the A,P and E sites lie in the large subunit, the sites are completed only when the small unit is present Dr Mark Carlile : Transcription & Translation 10 Transfer RNAs (tRNAs) tRNAs are adapter molecules that deliver amino acids to the ribosome The primary structure of a tRNA is between 60 and 95 nt long (most commonly 75 nt) that contains many (>20%) post-transcriptionally modified bases tRNA secondary structure: All tRNAs have the same basic secondary structure The secondary structure is called a clover-leaf (a) There are significant levels of intra-tRNA hydrogen bonding sites (b) Dr Mark Carlile : Transcription & Translation 11 Reading the genetic code The mRNA sequence carries its message in a code of 3-letter “words” The genetic code is essentially a continuous run of 3 nucleotide triplets known as codons Translation is the process whereby the codons are read and the information used to insert an amino-acid into the growing polypeptide Virtually every amino-acid is encoded by more than one codon – a redundancy is built-in to the code Figure 6-50 Molecular Biology of the Cell (© Garland Science 2008) Dr Mark Carlile : Transcription & Translation 12 Deciphering the genetic code: Nirenberg experiment Marshall Nirenberg (and colleagues) performed experiments wherein they synthesized mRNA with repeating nucleotides 5’-UUUUUUUU-3’-OH 5’-AAAAAAAA-3’-OH 5’-CCCCCCCC-3’-OH Poly-GGGGG was unstable (??) And added these to a test-tube containing an E.coli cell lysate (all the components needed for translation) They then isolated the resulting proteins and looked for which amino acids were incorporated 5’-UUUUUUUU-3’-OH :: gave poly-Phenylalanine 5’-AAAAAAAA-3’-OH :: gave poly-Lysine 5’-CCCCCCCC-3’-OH :: gave poly-Proline So some of the triplet codes were identified Dr Mark Carlile : Transcription & Translation 13 Deciphering the genetic code: Nirenberg experiment Marshall Nirenberg (and colleagues) performed experiments wherein they synthesized mRNA with repeating nucleotides More complex mRNAs where then generated: 5’-AUAUAU….. 3’-OH This gave a protein that contained alternating isoleucine and tyrosine So as to orientate the amino acid sequence they introduced UUU at the 5’-end 5’-UUUAUAUAU….. 3’-OH This gave Phenylalanine-Isoleucine-Tyrosine Orientating the sequence became very important once more complex mRNAs where generated Dr Mark Carlile : Transcription & Translation 14 Translation in prokaryotes The components required for translation include: mRNA, tRNA, ribosome, GTP, initiation factors and elongation factors As in transcription, translation is in 3 Stages Initiation Elongation Termination The movement of the ribosome along the mRNA is known as translocation Dr Mark Carlile : Transcription & Translation 15 Translation - initiation The purpose of the initiation step is to assemble the translation “machinery” at the translation start site The initiation complex is formed from the ribosome, mRNA and initiator tRNA (initiator tRNA is formylated methionine tRNA; it specifically recognises the AUG codon) The initiator tRNA enters the P-site – all subsequent tRNAs enter the A-site only In addition, 3 initiation factors and a molecule of GTP are required Dr Mark Carlile : Transcription & Translation 16 Translation - Elongation The elongation step involves Aminoacyl-tRNA delivery Peptide bond formation Translocation (movement) After initiation the P-site is occupied whilst the A-site is empty Three elongation factors are recruited to the initiation complex – all can bind GDP or GTP required to help deliver the aminoacyl-tRNAs (GTP hydrolysis causes relase of EF-Tu) Helps regenerate the release EF-Tu-GDP complex (Translocase): uses the energy from GTP hydrolysis to eject the tRNA from the P-site and move the peptidyltRNA into the P-site from the A-site. The ribosome complex maintains a 6 bp contact with the mRNA which stops frameshifting Elongation proceeds until a termination codon appears in the A-site Dr Mark Carlile : Transcription & Translation 17 Translation - Termination Termination is the process whereby the ribosome is dissociated from the mRNA There are no tRNAs that recognise a STOP codon Protein release factors interact with these codons and bring about the release of the polypeptide chain Release Factor 1 (RF1) recognises UAA and UAG RF 2 recognises UAA and UGA RF3 helps RF1 and RF2 to carryout their role The release factor causes the peptidyl transferase to transfer the polypeptide to water rather than the next tRNA – so the protein is then released EF-G and a RF are required for the disciation of the ribosome complex from the mRNA, and removal of the uncharged tRNA from the P-site Dr Mark Carlile : Transcription & Translation 18 Co-translational control: The tryptophan operon Remember that transcription and translation are carried out simultaneously in bacterial cells As an mRNA is generated it is also being “read” by a ribosome (protein production) The tryptophan operon uses this co-translation as a control point for gene expression regulation The trp operon, found in E. coli bacteria, is a group of genes that encode biosynthetic enzymes for the amino acid tryptophan – a rare amino acid The trp operon is expressed (turned "on") when tryptophan levels are low and repressed (turned "off") when they are high The trp operon is regulated by the trp repressor. When bound to tryptophan, the trp repressor blocks expression from the operon. Tryptophan biosynthesis is also regulated by attenuation (a mechanism based on coupling of transcription and translation). Dr Mark Carlile | Gene Expression (Part 1) 19 Co-translational control: The tryptophan operon Like in the lac operon – the tryptonphan repressor protein inhibits the expression of gene from the trp operon Attenuation is also used as a control mechanism: This mechanism is used when tryptophan levels are high and effectively stops the progression of the ribosome along the mRNA molecule Dr Mark Carlile | Gene Expression (Part 1) 20 Co-translational control: The tryptophan operon Attenuation is also used as a control mechanism: A control sequence is used to respond to tryptophan levels The control sequence lies between the operator and the first gene of the operon and is called the leader sequence The leader sequence encodes a short polypeptide and also contains an attenuator sequence. The attenuator does not encode a polypeptide, but when transcribed into mRNA, it has self-complementary sections and can form various hairpin structures. The attenuator polypeptide contains two tryptophan residues in it if there is NO tryptophan (i.e. the cell needs to generate more of it) then the ribosome stalls and an anti-terminator hairpin structure is generated = this allows transcription and translation to proceed The mRNA has discrete sequence sections that can selfassociate to generate hairpin structures Dr Mark Carlile | Gene Expression (Part 1) 21 Co-translational control: The tryptophan operon Attenuation is also used as a control mechanism: When there is lots of tryptophan you do not need to generate more The ribosome moves quickly along the mRNA and is then forced to stall and stop because a terminator hair-pin structure is formed Anti-terminator is generated using sequences 2 and 3 Terminator is generated using sequences 3 and 4 … on the emerging mRNA Dr Mark Carlile | Gene Expression (Part 1) 22 Interfering with protein translation Dr Mark Carlile : Transcription & Translation 23 Protein physicochemical properties Properties: Contains: carbon, hydrogen, oxygen, nitrogen Will have a defined structure/shape Will have a defined mass Will absorb light in the UV range Will contain both charged and uncharged amino acids Will contain hydrophilic and hydrophobic amino acids Dr Mark Carlile | DNA/RNA Protein in the Lab 24 Summary of this lecture: Today we have covered: mRNA Translation (protein synthesis) How the mRNA sequence is turned into protein How the genetic code was elucidated experimentally How transcription and translation can be combined for gene expression control How we can interfere with the translation process Dr Mark Carlile : Transcription & Translation 25