D 1.2 Protein Synthesis SL & HL

SL Notes 

Transcription: the synthesis of RNA using a DNA template 

·       Short lengths of DNA that codes for single proteins are called genes. 

·       The production of messenger RNA (mRNA) using DNA as a template is called transcription. 

·       Takes place in the nucleus of eukaryotes and cytoplasm of prokaryotes. 

Process of transcription 

·       Transcription occurs in three stages.  

·       In the initiation stage RNA polymerase binds to the DNA at the start of a gene and separates the two strands of the DNA by breaking the hydrogen bonds, exposing the bases.  

·       During the elongation stage, RNA polymerase builds a molecule of mRNA on the template or antisense strand. 

·       The other strand is known as the coding or sense strand.  

·       The RNA polymerase moves along the DNA reading it one base at a time, adding free RNA nucleotides to the growing mRNA.  

·       At the termination stage, a terminator sequence in the DNA is reached and the mRNA is released. The RNA polymerase detaches from the DNA strand, allowing the two strands to come together again. 

Hydrogen bonding and complementary base pairing 

·       RNA polymerase adds the free RNA nucleotides along the template strand based on the complementary base pairing rule.  

·       One of the differences between DNA and RNA is that DNA contains the base thymine while in RNA this is replaced with uracil . 

·       This means that the RNA polymerase will add uracil to the mRNA strand when it encounters adenine on the DNA template strand. 

·       It is only through the base pairing rule that the correct placement of bases, can be assured.  

·       When the correct RNA nucleotide is placed by RNA polymerase, it will temporarily form hydrogen bonds with the complementary base on the DNA template strand.  

·       Two hydrogen bonds form between A and T/U and three hydrogen bonds form between C and G. 

Stability of DNA templates 

·       The DNA within a cell is often transcribed many times and, for cells that do not go through regular cell divisions, the DNA needs to remain intact throughout the life of the cell.

·       If the DNA was to be degraded by the transcription process it would not be able to continue producing functioning proteins, which could stop the cell from functioning and even lead to cell death.

·       Sugar-phosphate backbone of DNA gives stability to the bases.

·       Hydrogen bonds between the 2 strands maintain the integrity of the molecule.

·       Single DNA strands are used as a template for transcription, without changing the base sequence.

Transcription as a process required for the expression of genes 

·       Many of our cells have the ability to switch genes on and off through the control of transcription.  

·       Once the mRNA is produced and it has migrated out of the nucleus into the cytoplasm in eukaryotic cells, the next step in protein synthesis can take place – translation. 

·       Almost every cell in our body has a complete copy of your DNA in its nucleus and therefore has the instructions to produce every protein required by your body.  

·       However, not all of our cells are making all proteins all of the time.  

·       That would be a tremendous waste of resources and energy.  

·       Being complex multicellular organisms, our cells are highly specialised and are designed to carry out specific functions, requiring only a specific number of the proteins found in our genetic code. 

Translation as the synthesis of polypeptides from mRNA 

·       The mRNA is read by the ribosome (either free in the cytoplasm or attached to ER) and using the code within the mRNA, amino acids are added in a specific sequence to form a polypeptide. 

·       The sequence in which amino acids are assembled determines the polypeptide produced. 

·       Therefore, gene expression (completed during translation on ribosomes) is a function of complementary base pairing in nucleic acids. 

Roles of mRNA, ribosomes and tRNA in translation 

·       The mRNA brings the code from the DNA in the nucleus in its base sequence which has the instructions for the polypeptide to be produced. 

·       The site of translation is the ribosome.  

·       The structure of the ribosome brings the mRNA and the tRNA together in the correct orientation so that translation can occur efficiently and correctly. 

·       Ribosomes are very complex structures, consisting of proteins and ribosomal RNA molecules.  

·       Ribosomes have a small and a large subunit, with three binding sites for tRNA molecules. 

·       The mRNA binds to the small subunit, while up to two tRNAs can bind to the large subunit of the ribosome at a time. 

·       tRNA is a single-stranded RNA molecule that folds on itself to form a cloverleaf-shaped structure with double-stranded regions and three hairpin loops.  

·       Each tRNA has a specific corresponding amino acid attached to it through the process called amino acid activation to form a tRNA-amino acid complex. 

·       ATP and enzymes specific to each amino acid are used for amino acid activation. 

·       The specificity of enzyme ensure that correct amino acids are used in the right sequence. 

·       A tRNA recognises and binds to its corresponding codon on the mRNA in the ribosome, using its anticodon end. 

·       When 2 such anticodons of tRNA-amino acid complex are temporarily bonded to the mRNA codons on the ribosome, a peptide bond is formed between adjacent amino acids. 

·       The first tRNA is freed which moves into cytoplasm for reuse. 

·       The ribosome moves on to the next mRNA codon. 

·       The ribosome continues to move codon by codon until it reaches a stop codon. 

·       Consecutive amino acids link together by condensation reactions forming peptide bonds, which eventually emerges from the larger subunit. 

Complementary base pairing between tRNA and mRNA 

·       Placement of the correct amino acids in the correct sequence to produce a functional polypeptide is made possible by continuation of the complementary base pairing rule. 

·       The pairing is between the bases in the mRNA and those found on the tRNA molecules that are carrying the amino acids. 

·       The code found in the mRNA is read in groups of three. This triplet code is known as a codon.  

·       A codon can be any three RNA bases in a sequence, such as UAG or CCA.  

·       Each of these codons is the code for the placement of a specific amino acid.  

·       The tRNA molecules, each carrying their specific amino acid, have their own three-base code called anticodon that is complementary to the matching codon on the mRNA.  

·       The complementary nature of the codon and anticodon ensures that the correct amino acid is placed in sequence  

Growing polypeptide chain 

·       When the elongation process begins, the ribosome begins to move along the mRNA, one codon at a time. 

·        As each codon is brought into place, a new tRNA carrying the corresponding amino acid, attaches and moves previous tRNA molecules to the next position. 

·       As new amino acids are delivered, condensation reactions are catalysed and peptide bonds are formed between them. 

·       When these steps are constantly repeated a polypeptide chain is elongated eventually emerging from the larger subunit. 

Features of the genetic code 

·       The genetic code is broken down into codons found on the mRNA.  

·       Each codon represents a specific amino acid.  

·       As there are four bases in RNA and they are arranged in groups of three, this means there are 64 different possible combinations (4 ). 

·       There are 20 amino acids in total and, as 64 codons can be formed by using the four bases, some amino acids are coded for by more than one codon, accounting for the degeneracy of the genetic code.  

·       There are also specific codons that act as a signal for the protein translation machinery to start (AUG) or to stop (UAG, UGA, UAA). 

·       Another important feature of the genetic code is that it is universal.  

The same codons, code for the same amino acids in every organism on Earth with very few exceptions. 

·       This is evidence that all existing life on Earth has likely descended from a common ancestor.  

Mutations that change protein structure :

·       In a point mutation, a single nucleotide is changed.  

·       It may be deleted, added or replaced with another.  

·       This usually happens during DNA replication but can also occur during transcription. 

·       Due to the degeneracy of the genetic code, sometimes a change in third base produces a codon which codes for the same amino acid, and there is therefore, no effect. 

·       This is essentially a silent mutation.  

·       If the point mutation occurs to the first or second base in a codon there is a higher chance that it will lead to the addition of a different amino acid in the sequence for that polypeptide.  

·       If the mutation changed a codon to a stop codon, that would end the polypeptide early and it would be unlikely to function as required.  

·       A change to another amino acid would likely lead to an effect on the overall polypeptide. 

Example of point mutation:

·       Sickle cell anaemia resulting in 6^th amino acid glutamate of beta globin chain getting replaced by valine.  

·       Such haemoglobin called Haemoglobin S is hydrophobic attracting other Hb molecules to stick to it. 

·       In areas with low pO_2, sickle cell Hb clumps to form long fibers which blocks blood capillaries preventing circulation of normal red blood cells.   

·       This results in people suffering from anaemia - inadequate delivery of oxygen to cells.   

·       People with single allele for Haemoglobin S is said to have sickle cell trait – mild anaemia.

·       People with double alleles for Haemoglobin S is said to have sickle cell anaemia which is a serious condition. 

D1.2 Protein Synthesis  

HL notes 

Directionality of transcription and translation:

·       RNA polymerase builds mRNA molecules in a 5′ to 3′ direction.  

·       RNA polymerase can catalyse the formation of a phosphodiester bond between the 3′ end of one RNA nucleotide and the 5′ end of the next nucleotide. 

·       During translation the mRNA moves through a ribosome in a 5′ to 3′ direction as the codons on it are read to assemble the amino acids in the correct sequence.  

·       This is because the 5′ end of mRNA only fits the binding site of the ribosome in correct orientation.   

·       If the mRNA was able to attach in either direction and the code was read backwards, the code would be completely different, resulting in a completely different polypeptide. 

Initiation of transcription at the promoter 

·       On the DNA, just before a gene, is a region of code known as the promoter 

·       At the promoter, proteins known as transcription factors can bind.  

·       It is the binding of the correct transcription factors in the correct orientation that allows the RNA polymerase to also bind and then begin to transcribe the DNA into RNA.  

·       If those transcription factors are missing or something has blocked their ability to bind to the promoter, transcription will not take place and that gene cannot be expressed. 

Enhancers 

·       Specific transcription factors bind at the enhancer to increase (or decrease) the rate of transcription. 

·       Enhancers are located upstream of the promoter and gene sequence. 

·       Binding of activator proteins to the enhancer site forms a new complex that can contact the promoter-transcription factor complex.   

·       This increases the rate of transcription. 

Non-coding sequences in DNA do not code for polypeptides:

·       Regulators of gene expression: these are DNA sequences that regulate gene expression in various ways. Promoters act as a binding point for the RNA polymerase enzymes that catalyse the transcription process. Enhancers or silencers on the DNA may act as binding sites for proteins that either increase or decrease the rate of transcription respectively. 

·       Introns: these are DNA base sequences found within eukaryotic genes that get removed at the end of transcription. They do not contribute to the amino acid sequence of the polypeptide made from the gene. 

·       Genes for tRNAs and rRNAs: These genes code for RNA molecules that do not get translated into proteins, but instead fold to form tRNA molecules that play an important role in translation or the rRNA that forms part of the structure of ribosomes. 

·       Telomeres: are repetitive sequences that protect the ends of the chromosome by binding with proteins.  Telomeres help ensure that DNA is replicated correctly.   Prevents chromosomal ends from attaching to each other which may activate the cell’s system for monitoring DNA damage, therefore avoiding apoptosis. 

·       Major lengths of non-coding DNA: are the VNTR’s which are clusters of repetitive sequences of DNA. 

Post-transcriptional modification in eukaryotic cells 

·       In eukaryotes, the immediate product of an mRNA transcript is called pre-mRNA which needs to be modified to form mature mRNA 

·       Three post-transcriptional events must occur 

o   A methylated cap is added to the 5' end to protect against degradation by exonucleases 

o   A poly-A tail (long chain of adenine nucleotides) is added to the 3' end for further protection and to help the transcript exit the nucleus 

o   Non-coding sequences (introns) are removed and coding sequences (exons) are joined together 

Polyadenylation and 5′ capping

The 3′ end of the pre-mRNA is cleaved to free a 3′ hydroxyl group. 

An enzyme poly A-polymerase adds a chain of adenine nucleotides to the RNA. 

This process is called polyadenylation. 

The poly-A tail is about 100-250 nucleotides long. 

The 5′ is capped by the addition of a 5′ modified guanine nucleotide. 

A mature mRNA is formed that will be protected from enzymatic degradation and can be exported out of the nucleus into the cytoplasm for translation. 

RNA splicing 

·       Introns that are non-coding sequences of DNA are removed by a spliceosome (a large RNA protein complex with enzymatic properties) 

·       Spliceosome is also controlled by a gene. 

·       Pre-mRNA introns are spliced out using spliceosome. 

·       It recognizes a highly conserved region (splice site) between the 3′ end of the exon and 5′ end of the intron and cleave the phosphodiester bond between the nucleotides. 

·       All exons are joined together to form one polypeptide from one gene. 

Alternative Splicing 

·       Sometimes an mRNA can be spliced in multiple ways by combining different exons and omitting others.  

·       This results in different versions of proteins which will often function differently.

·       The number of proteins produced can be greater than the number of genes present. 

·       This explains why the human genome consists of the same low number of genes as some small invertebrates. 

Initiation of translation – Activation of amino acids 

·       Each amino acid is linked to a specific tRNA before it can be used in protein synthesis. 

·       This is catalysed by the action of a tRNA activating enzyme. 

·       There are 20 different tRNA activating enzymes, one for each of the 20 amino acids. 

Initiation of translation 

·       The 5′ terminal of the mRNA binds to the small ribosomal subunit. 

·       The ribosome then moves along the mRNA until it finds the start codon AUG.  

·       The anticodon of the initiator tRNA, carrying the amino acid methionine, binds to the codon of the mRNA.  

·       Finally, the large ribosomal subunit joins to complete the assembly of the translation complex. 

Elongation of polypeptide chain 

·       The initiator tRNA currently occupies the “P” site,  

·       The next codon on the mRNA signals for the corresponding tRNA to bind at the “A” site 

·       The two amino acids (attached to the tRNAs) are linked with a peptide bond, forming a dipeptide. 

·       The ribosome shifts along the mRNA by one codon (three bases) at a time. 

·       The initiator tRNA in the “P” site moves to the “E” site which releases it. 

·       The tRNA carrying the dipeptide moves from the “A” site to the “P” site. 

·       The next mRNA codon is exposed and a tRNA with the complementary anticodon binds to the unoccupied “A” site whilst the third amino acid is linked to the dipeptide. 

·       In the cytoplasm, free tRNA molecules bind to their corresponding amino acids and transport them to the ribosome. 

·       The cyclical process is repeated as new amino acids are added to the growing chain. 

Termination 

·       Translation stops when the ribosome encounters a STOP codon on the mRNA (UAA, UGA, UAG). 

·       The completed polypeptide is released from the ribosome into the cytoplasm. 

·       The ribosomal subunits separate. 

·       The mRNA is released. 

Modification of polypeptides into their functional state 

·       After a polypeptide is synthesised at a ribosome by translation, it is often still not in its final functional state.  

·       This requires further modification of the polypeptide.  

·       When polypeptides are synthesised by ribosomes on the rough endoplasmic reticulum, they are packaged in vesicles which carry them to the Golgi apparatus.  

·       It is in the Golgi apparatus where many of these modifications are carried out. 

Post-translational modification of pre-proinsulin to insulin 

·       Insulin is a peptide-based hormone produced in the beta cells of the pancreas. 

·       When the insulin gene is translated, the product is pre-proinsulin.  

·       This is a polypeptide 110 amino acids in length.  

·       It is composed of four main sections: a signal peptide (28 amino acids), an A chain (21 amino acids), a B chain (30 amino acids) and a C-peptide (31 amino acids).  

·       Once the pre-proinsulin enters the rough endoplasmic reticulum, the signal peptide is removed.  

·       The remaining polypeptide is now called proinsulin.  

·       The proinsulin is packaged into vesicles that move to the Golgi apparatus where Disulfide bridges form between the A chain and the B chain.  

·       The C-peptide is removed and mature insulin remains. 

Recycling of amino acids by proteasomes 

·       The proteome is the total of all proteins made and used by the body. 

·       The proteome of an organism is maintained to ensure optimal functioning of the body. 

·       This is done by proteolysis carried out by proteasomes. 

·       Proteasomes are protein complexes that degrade unneeded, misfolded or damaged proteins. 

·       In this way, recycling of existing amino acids take place in addition to dietary supply to ensure sufficient turnover. 

Mechanism of protein degradation by proteasome 

·       A short chain of regulatory protein called ubiquitin binds the protein to be degraded. 

·       The marked protein binds to the proteasome at the cap. 

·       The protein is fed into the core particle where it is broken into peptides. 

·       The peptides are further processed to form a pool of amino acids from which new proteins can be synthesized.