Lecture 4 - Week 4 Protein Synthesis (BI1CMP1)

From nucleic acids to proteins – the genetic code and translation BI1CMP1 Dr Susanna Cogo [email protected] Intended Learning Outcomes (ILOs) At the end of this lecture, you will be able to: Discuss the structure and functions of proteins Explain the genetic code Describe t...

From nucleic acids to proteins – the genetic code and translation BI1CMP1 Dr Susanna Cogo [email protected] Intended Learning Outcomes (ILOs) At the end of this lecture, you will be able to: Discuss the structure and functions of proteins Explain the genetic code Describe the structure and function of tRNA and rRNA Outline the main steps of translation From nucleic acid to protein Replication Translation is the process by which a protein is synthesized from the information contained in a molecule of messenger RNA (mRNA). Proteins have many different functions Enzymatic Structural Proteins Regulatory Transport Can you think of an example? Proteins have many different functions Enzymatic Structural Proteins Regulatory Transport Aminoacids (aa) are the monomers of proteins https://www.youtube.com/watch?v=BLj-7pYAyws Aminoacids are the monomers of proteins Polypeptide chains are formed via peptide bonds The sequence of aminoacids in a protein represents its primary structure From 2D to 3D Interaction with neighbouring aminoacids leads to folding into a secondary structure Interaction of secondary structures leads to a 3D conformation: tertiary structure Association of multiple polypeptide chains represents the quaternary structure Proteins can also contain discrete domains = functional units From nucleotides to aminoacids – the genetic code The basic unit of the genetic code is called codon 1961: Crick and colleagues specify that each codon is made of three nucleotides The flow of information from gene to protein is based on a triplet code: a series of three-nucleotides Breaking the genetic code Codon: a triplet RNA code 64 possible codons: ‒ 3 stop codons ‒ 61 sense codons (termination codon) All 64 codons were deciphered by the mid-1960s The genetic code is “degenerate” (more than one codon may specify a particular amino acid) but not ambiguous (no codon specifies more than one amino acid) Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced Breaking the genetic code The codons are written 5′→3′, as they appear in the mRNA. AUG is an initiation codon; UAA, UAG, and UGA are termination (stop) codons https://www.youtube.com/watch?v=QLhIg60hK9M The genetic code is (almost) universal The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals Genes can be transcribed and translated after being transferred from one species to another Three main RNA classes are involved in protein synthesis Transfer RNAs Transfer RNA (tRNA) is the link between the genetic code and the amino acids that make up a protein The role of tRNA is to transport a specific amino acid to the ribosome Each tRNA is only able to bind one amino acid (and can be identified by specific nomenclature, e.g. tRNAPro is the tRNA bound with proline) Each tRNA is encoded by a specific gene (or usually multiple gene copies) More than the 4 “classical” bases are present in tRNA, arising from tRNA-modifying enzymes How many tRNA do you predict there are? The structure of transfer RNAs 74-95 nt long Shared CCA sequence DHU T𝛙C tRNAs are processed after transcription Ribosomal RNA (rRNA) 26S in yeast 28S in humans Ribosomes are complexes made up of more than 50 RNA molecules and proteins Ribosomes are the location of translation from mRNA to proteins Very abundant (>20,000/cell) – they contain approximately 80% of the total RNA in a cell “S” (Svedberg) is a unit of size based on the speed of centrifugation – note that it is not additive!!! Eukaryotic ribosomal RNA (rRNA) rRNA genes are also present in multiple copies, which in eukaryotes are clustered 2 rRNA genes:  large (coding for 28S, 18S and 5.8S) – processed post transcription!  small (5S) Let’s translate! What? When? Where? Let’s translate! What? mRNA When? After mRNA transport from nucleus to cytoplasm Where? Ribosomes Ribosomes bind near the 5’ of mRNAs Synthesis begins at the amino end of the protein (N-terminus), and new residues are added at the carboxyl end (C-terminus) Interactions happen among RNA families The stages of translation 1) tRNA charging: binding of tRNAs to amino acids 2) Initiation: assembly of the machinery at the ribosome 3) Elongation of the polypeptide chain through addition of new amino acids at the C-ter 4) Termination: protein synthesis ends at the stop codon and the machinery is released tRNA charging The CCA sequence is shared by all tRNAs The carboxyl group of the amino acid is attached to the nitrogenous base of A at the 3’ end of tRNA Specificity is determined by aminoacyl-tRNA synthetases tRNA charging Recognition of tRNA by the aminoacyl-tRNA synthetases is mediated by the nucleotide sequence Recognition of amino acid by the aminoacyl-tRNA synthetases is mediated by size, charge and R groups tRNA charging Key residues in the recognition Aminoacyl-tRNA synthetases have proofreading activity Initiation toolkit Assembly of the components required for protein synthesis: 1) mRNA 2) Small and large subunits of the ribosome 3) Initiation factors (only 3 in prokaryotes) 4) Initiator tRNA (Met-tRNAiMet) 5) GTP Initiation stages 3 main stages: 1) mRNA binds the small subunit of the ribosome 2) Initiator tRNA binds to the mRNA (anticodon-codon binding) 3) The large ribosomal subunit joins the complex Initiation of translation (i) The small (SSU) and large (LSU) ribosomal subunits need to be separate for the mRNA to bind the small subunit A 43S pre-initiation complex composed of SSU, Met-tRNAiMet and initiation factors recognise and bind the 5’ cap in the mRNA (different compared with bacteria!!!) The 43S pre-initiation complex scans the mRNA until the first AUG codon is found AUG is surrounded by a consensus sequence which helps the recognition: the Kozak sequence (ACCAUGG) After recognition, codon and anticodon (in the tRNA) bind Initiation factors are released LSU binds the complex Initiation of translation (ii) At least 12 IFs (eIFs) required which mediate  Preventing binding of LSU via binding to SSU  Recognition and binding of the 5’ cap  Recruitment of the initiator tRNA  Binding between the initiator tRNA and the initiator codon  LSU binding The pioneer round of translation CBC (cap-binding complex) promotes the nucleus to cytoplasm export and a pioneer round of translation to check for errors CBC is then substituted with eIF-4E for continuation The poly-A is also involved in the initial phase: interaction with the 5’ cap via regulatory proteins and formation of a closed loop – this promotes stabilization of the SSU/mRNA binding Exceptions:  no need of the 5’ cap  different initiator codons  uORFs: short reading frames upstream of AUG (in the 5’ UTR) that can be translated into proteins Elongation Requires:  The 80S initiation complex  Charged tRNA (= with aa)  Elongation factors  GTP A ribosome has 3 possible tRNA binding sites:  Aminoacyl (A)  Peptidyl (P)  Exit (E) The initiator tRNA occupies the P site, the others occupy the A site first At the end of translation initiation, the ribosome is bound to mRNA and the initiator tRNA is bound to AUG in mRNA Elongation Elongation happens in 3 steps: 1) Binding of a charged tRNA to the A site (mediated by elongation factor eEF1a binding to GTP and the charged tRNA), followed by tRNA-mRNA pairing and release of GDP- eEF1a. Other eEFs mediate GDP-GTP conversion. 2) Formation of a peptide bond between amino acids in the A and P sites, leading to release of the aa in the P site from tRNA. This happens in LSU and is mediated by 28S rRNA (ribozyme). 3) Translocation: movement of the ribosome in 5’ to 3’ direction, mediated by eEF2 (with GTP hydrolysis). tRNAs do not move because they are still bound to mRNA. tRNA moves from P to E and is released into the cytoplasm to be recharged. The cycle repeats, with the polypeptide sequence remaining attached to the P site. If the mRNA forms secondary structures, helicase activity in the SSU unwinds them. Termination Protein synthesis ends when a termination codon is reached No tRNA for the termination codon, hence the A site is left empty Release factors (RFs) mediate the final steps: 1) eRF1 is required for recognition of the stop codon 2) eRF3 supports the cleavage of the tRNA-polypeptide bond in a GTP dependent manner 3) Other RFs mediate the release of tRNA and mRNA and the ribosome dissociation https://www.youtube.com/watch? v=qIwrhUrvX-k Is the termination codon the (3’) end of the mRNA? Protein synthesis and disease Protein synthesis and disease Exceptions to the central dogma of molecular biology Rufai, Gupta & Singh, 2020 https://www.youtube.com/watch?v=d2daVaZZHYE