1€¦ · Web viewPopulation studies: Analysis of human or other species’ population genetics can be rapidly performed using PCR analysis. Sequencing: DNA sequence analysis previously

1.1 IntroductionIn this Topic the structure of DNA will be described and the DNA replication explained.

(a) Chromosomes Chromosomes threadlike structures located in the nucleus of all cells (except red blood

cells). Chromosomes contain the genetic material that determines the characteristics of a living

thing. All cells store their genetic information within chromosomes in the base sequence of DNA

(b) Genes Gene: Sequence of 3 bases on the DNA.

Genotype (pattern of genes on a chromosome) is determined by the sequence of bases.

Phenotype is determined by the proteins made by the instructions encoded on DNA.

DNA chemical sequence is unique to all individuals (except identical twins). DNA can be cut up and separated, forming a sort of individual 'bar code'. This is why people can be identified using DNA fingerprinting.

(c) DNA Deoxyribonucleic acid or DNA codes for proteins in the cell. During cell division DNA must be replicated so that daughter cells carry an identical set of

instructions to those in the parent cell. DNA molecules are large and complex. DNA is inherited.

QUESTIONSQ1: Give another name for the type of cell division described in part (c) DNA.

Q2: State where within chromosomes all cells store their genetic information.

Q3: How does the genetic information on the DNA determine the phenotype?

Q4: Which organelle in the cell contains DNA?a) Golgi apparatusb) Endoplasmic reticulumc) Lysosomed) Nucleus

Q5: DNA is a component of which structures?

1.2 DNA Structure and replication

DNA STRUCTURE

DNA is an example of a ______________ acid. DNA is a long chemical sequence and this sequence contains the information needed for that living thing to develop, survive and pass its genetic information on to the next generation.

DNA is a DOUBLE HELIX characterised by the following:

(I) Nucleotides Nucleotides are organic molecules that act as monomers  (or subunits) of nucleic acids  like DNA.

Nucleic acids like DNA are made from repeating unitscalled __________________________.

NUCLEOTIDES are the building blocks of nucleic acids and are composed of:

- nitrogenous base  , - deoxyribose   sugar (five-carbon sugar) - phosphate group  

Activity: COMPLETE THE NUCLEOTIDE DIAGRAM YOU MUST BE ABLE TO DRAW A NUCLEOTIDE IN THE EXAM!

(II) Sugar–phosphate backboneNucleotides join together to form chains.The bonds between nucleotide are called strong deoxyribose sugar-phosphate bonds.

When a chain of nucleotides join through sugar phosphate bonds this forms the deoxyribose sugar-phosphate backbone.

Activity: 1. Draw the strong deoxyribose-phosphate

bonds between the nucleotides.

2. Write 3’ and 5’ at the correct end of the strand.

( III) HYDROGEN BONDS between nitrogenous bases

DNA encodes hereditary information in a chemical language.

There are 4 nitrogenous bases: Adenine, Thymine, Cytosine and Guanine.

(IV) Base pairing is specific in the pattern:

Adenine ====== Thymine Guanine ====== Cytosine

(V) Double stranded antiparallel structure:

DNA is composed of 2 polynucleotide chains

The two polynucleotide chains run antiparallel with a deoxyribose sugar at the 3’ end and phosphate group at the 5’

(VI) Each chain is joins to a COMPLIMENTARY chain by weak HYDROGEN BONDS between nitrogenous bases. The DNA structure is a antiparallel stranded double helix.

The four bases in DNA. The complimentary bases are: ____________ and ____________, ____________ and ______________. It is only the bases which makes one nucleotide different from another. Since there are only four different bases, there can only be four different _________________ in DNA . Nucleotides join together by ______________ bonds to form a sugar-phosphate backbone. The bases pair off and are held together by weak _______________ bonds, forming a twisted double ___________. Adenine is always opposite ____________ ; ____________ is always opposite cytosine. The paired antiparallel strands coil forming a _______________ _______________.

ACTIVITIESLearner activity 1: DNA – True or False

Learner activity 2: Short questions Create your own DNA! Using cutting and pasting

Learner activity 3: DNA extraction. Collect a worksheet.

Learner activity 4: Discovering the structure of DNA. Collect a summary sheet.

Learner activity 5: How the structure of DNA was discovered. Case study.

Learner activity 6: Answer question set B

Learner activity 1: TRUE or FALSE1. DNA is found in some living cells

2. DNA is the heredity material – it is passed on from one generation to the next.

3. Your DNA is the same as the DNA of the person sitting next to you.

4. DNA is found in the cytoplasm of the cell.

5. The DNA sequence – the chemical language – doesn’t matter as it doesn’t change anything about the living thing.

Learner activity 2: SHORT QUESTIONS1. What is a "strand" of DNA?

2. How many strands make up a DNA double helix?

Web site http://www.youtube.com/watch?v=BmDG_fKUTR8

3. Each strand is made up of two zones or regions. One zone of each strand is made up of …identical repeating units, while another zone is made up of differing units. …What are the zones of each strand called?

4. The DNA double helix looks like a twisted ladder. What makes up each rung of the ladder?

5. What holds the rungs together at the sides?

6. What holds one strand against the other in the double helix?

7. What are the four pairs of DNA bases that form in the double helix?

8. How can A distinguish T from C?

9. Which DNA double helix do you think would be harder to separate into two strands: DNA ……...composed predominantly of AT base pairs, or of GC base pairs? Why?

10. What is a mutation?

DNA HISTORY FOR CASE STUDYAt the turn of the 20th century scientists new that chromosomes contained the ‘genetic material’ that enabled organisms to pass on hereditary material or ‘genes’ to the next generation. Over the next 50 years six scientists research elucidation of the structure of DNA and reveal the following: . Research conclusion: Scientist(s) .

1. DNA that was the ‘genetic material’, Griffiths and Avery et al.,2. Nucleic acid bases of DNA were found in set ratios, Chargaff3. DNA was a double helix, composed of paired Franklin & Wilkin : Watson & Crick

bases along two sugar phosphate backbones,running parallel but in opposite directions

Over the next 30 years further research proved 1. ‘genetic material’ was composed of: 1.DNA with protein, 2. RNA 2. The DNA made into protein using RNA as a messenger. 3. There is a vast number of possible amino acid combinations in proteins as:

a. The 4 bases in DNA can be arranged in any combination b. The 20 amino acids which can be arranged in any order.

RESEARCH HISTORY FOR CASE STUDYGriffiths: Experimental evidence from Bacterial transformationConclusion: A material from a virus which was heat stable and not protein could be transformed ………………….into and between mice.

Fig.1. Griffith’s experiment showing the fate of mice when infected with different strains or strain combinations of the Streptococcus pneumonia virus. The experiment in the final column revealed to Griffith’s that there exists a ‘transforming principle ‘which he assumed to be protein.

Avery et al.,: Experimental evidence to identify the transforming agent. Conclusion: Avery deduced which of the macromolecules (polysaccharide, protein, RNA or DNA) were transformed from bacteria to mice. He used a series of systematic enzyme assays to degrade each of the macromolecules one at a time. They discovered that it was only through the use of DNAse (which degrades DNA) did the transformation cease.

Hershey & Chase: Phage experiments

Experiment 1, on the top line, shows the results of when DN radioactively labelled, with radioactivity being detected in the cell. Experiment 2, shows the results when protein was radioactively labelled, with radioactivity being detected in the solution which

contained the ‘phage ghosts’. The results revealed that DNA was the genetic material.

Erwin Chargaff - Chargaff’s Rules and the base composition of DNAChargaff analysis showed:

1. Hydrolysed DNA from a number of different organisms and found that DNA always contained 50% A & T bases and 50% G + T bases.

2. Number of adenine + thymine basis equal Number of guanine + cytosine. 3. The percentage of each base was species specific and responsible for variation within a species. The bases were not arranged in the same pattern on DNA across all species, there was the possibility of DNA holding the key to the diversity of life.

Example: What is the percentage of Cytosine in DNA if the percentage of Adenine is 20%?Percentage of A = Percentage of T Thus, % T = 20%Percentage A+ T = Percentage G + C Thus, % G + C = 80% Percentage of G = Percentage of C Thus, % G = 40%

Rosalind Franklin & Maurice Wilkins–DNA is helical & has measured regularities in its structureUsing a technique called x-ray diffraction, Franklin and Wilkins produced x-rays of molecular structure of DNA. The technique involved sending beams of parallel x-rays at isolated strands of DNA. When the x- rays collide with the atoms they diffract to an extent which is dependent on the molecular weight and spatial arrangement of the atoms. These diffracted x-rays are then collected on a photographic plate and the resulting pattern analysed.

James Watson & Francis Crick–Model for physical & chemical structure of DNAWatson and Crick came to their conclusions from the experimental data of others, (Chargaff, Wilkins and Franklin), and offered no fresh experimental data of their own.

The structure of DNA: DNA is a double helix formed from two polynucleotide

chains that are wound around in a clockwise direction.

1

2

The two polynucleotide chains are antiparallel, meaning that they are positioned head to tail (each chain has a 5’ end and a 3’ end and so the 5’ end of one lies opposite the 3’ end of the other)

The sugar-phosphate backbone which forms from the connection between the nucleotides is on the outside of the double helix. The nucleotide bases are on the inside, perpendicular to the sugar phosphate backbone.

Each base is connected to a complementary base on the parallel chain by weak hydrogen bonds. Adenine always pairs with thymine by two hydrogen bonds and guanine to cytosine by three hydrogen bonds. No other base pairings would work to give rise to this DNA structure.

Structurally, the base pairs are 0.34 nm apart and each 3600 degree turn of the helix is 3.4 nm (10 base pairs). Also, as the nucleotides are unequally spaced between the two polynucleotide strands because they are antiparallel, there are unequal grooves in the helical twist.

1(a)(ii) Organisation of DNA in prokaryotes and eukaryotesIn both prokaryote cells and eukaryote cells DNA is organised into structures called chromosomes.

CELL TYPES

Prokaryotic cells Eukaryotic cellsExamples of cells: 1. Bacteria and fungal cells 1. Animal and Plant cells

Nucleus present: 2. No nucleus 2. Contain a nucleus

Chromosome number: 3. One 3. Numerous

Chromosome shape: 4. Circular 4. Linear

DNA folding type 5. Supercoiled DNA 5. DNA coiled with proteins

Additional DNA: 6. In plasmids 6. In mitochondria and/or chloroplasts

Prokaryotic chromosomesPROKARYOTE CHROMOSOME: Prokaryotes have a single double-stranded and circular chromosome, which carries the organism’s genetic material. In bacteria, DNA is packaged tightly, into a given area of the cell called the nucleoid.PROKARYOTE PLASMID: If the prokaryote has a second circular chromosome carrying a DNA sequence that is superfluous to the existence of the organism then it is called a plasmid. Some prokaryotes, have more than plasmid, which can be either a copy of the first or be composed of a different DNA sequence and which can contain essential or non-essential genes.

(A) Circular chromosomal DNA and plasmids in bacteria. (B) Circular plasmids in yeast.

SUPERCOILING Supercoiling is the key way in which prokaryotes package the huge length of DNA that makes up their genome into a small area in the cell. Imagine an elastic band being twisted, it first makes twists which then produce an ever-tighter wound band. The elastic band will finally begin to supercoil, producing bends of twisted elastic, making the elastic band ever more compact. This happens when DNA supercoils. Not super coiled Super coiled

Further information about chromosomes in prokaryotes can be found at the Nature Scitable site: http://www.nature.com/scitable/topicpage/genome-packaging-in-prokaryotes-the-circular-chromosome-9113

Eukaryotic chromosomesIn contrast to prokaryote chromosomes:1. Eukaryotes have several linear chromosomes contained within a membrane-bound nucleus. 2. The number of eukaryotic chromosomes is consistent in a species but can vary across species.e.g.Humans have a haploid set of 23 chromosomes; wheat has a haploid set of 7 chromosomes.

DNA in eukaryotes:Eukaryotes use different methods from Prokaryotes to compact their DNA into chromosomes.

Linear chromosomes in the nucleus. Circular chromosomes in

mitochondria/chloroplasts

Eukaryotic cells also contain extra packages of DNA out with the nucleus: Mitochondrial DNA mtDNA is found in both plants and animals, whereas chloroplast DNA (cpDNA) is only found in green plants. These genomes are not inherited in a medallion fashion like chromosomes in the nucleus, but instead are inherited solely from the mother with the other cytoplasmic organelles.

MITOCHONDRIAL DNA (mtDNA)MtDNA is often circular, double-stranded and lacking in the structural proteins of the nuclear chromosomes, much like the chromosomes found in prokaryotes. Their size varies enormously between species. In the main, the mitochondrial genome codes for transfer RNAs (tRNAs), ribosomal RNAs (rRNAs) and some subunits of proteins found in mitochondria.

CHLOROPLAST DNA (cpDNA)CpDNA is structured similarly to mtDNA: it is circular, double-stranded and lacks structural proteins. Chloroplasts can contain many copies of cpDNA, with the copy number variable between species. The size of the cpDNA genome varies between species and can be anywhere between 80 and 600 kb. Amongst the genes on the cpDNA lie those that code for the rRNAs, tRNAs and some proteins required for translation, transcription and photosynthesis.

(The origin of mitochondria and chloroplasts is debated scientifically. The endosymbiont theory, proposes both mitochondria and chloroplasts were originally free-living bacteria that invaded primitive eukaryotic cells and through the course of evolution the bacteria and the

original host became dependent to the point where one could not live without the other. This emphasises the interconnectivity of living things).

Packaging of DNA in eukaryotic chromosomes

The organisation of DNA in a eukaryotic cell depends on the stage of mitosis they are in.

The level of organisation in the packaging of DNA is truly amazing.

The length of a DNA molecule, if held taut, end-to-end, in just one human chromosome would measure 4 cm. Given that cells are not nearly that big, our cells are capable of packaging this amount of DNA into chromosomes 1.2–2 µm in length. (This means that end-to-end you could fit 10,000 chromosomes along the length of a fingernail).

If you take this figure and the fact that we have 46 chromosomes in each cell we can calculate a total length of DNA in one human cell to be 1.84 m (the height of a 6-foot person). Considering the number of cells we have, we have enough DNA that if put end to end it would reach the moon and back!

Figure 4: Overview of the levels of packaging seen in a metaphase chromosome

Nucleosomes

DNA double helix is wrapped around histone proteins forming nucleosomes (beads on a string)

The pieces of DNA between the nucleosomes is known as linker DNA and is a constant length.The combination of DNA and protein is called chromatin.This level of organisation is seen throughout the cell cycle and mitosis.

DNA in the linear chromosomes in the nucleus of eukaryotes is tightly coiled and packaged with associated proteins.

There are 4 levels of packaging in cells, the highest of which is only seen during metaphase.

Level 1: Nucleosomes

Level 2: Thick chromatin fibre

Level 3: Looped fibres

Level 4: More folds to make the most compacted chromosome

DNA in the form of a double helix is wound around histone proteins, forming nucleosomes. Histones are positively charged and so bind tightly to the negatively charged DNA. The lengths of DNA between the nucleosomes are called linker DNA. This level of organisation is seen throughout the cell cycle, with only transient separation during replication.

When several nucleosomes form this is commonly called ‘beads on a string’.The combination of proteins and DNA is called chromatin, so the beads on a string structure shown here is a chromatin fibre.

The length of nucleosomes then coils to form a thicker chromatin fibre, about 30 nm thick, due to interactions between the nucleosomes and linker DNA. This level of packaging can be seen during interphase.

The thick chromatin fibre then folds along a non-histone protein scaffold, producing fibres that are now 300 nm thick. This level of packaging can be seen during prophase.

The chromatin (DNA + protein) folded along the protein scaffold then folds further to produce the compacted chromosomes that are seen during metaphase. This is DNA in its most compacted form. Note that this image shows a metaphase chromosome, which consists of two chromatids following replication.

When several nucleosomes form this is commonly called ‘beads on a string’.The combination of proteins and DNA is called chromatin, so the beads on a string structure shown here is a chromatin fibre.

Arrangement of DNA in a Prokaryote and Eukaryote cell

DNA is packaged in a different way in eukaryotes and prokaryotes.Prokaryote Eukaryote Both

Student activity 9: Prokaryote or eukaryote? Students cut out the statements on the sheets and place them into one of three groups: prokaryotes, eukaryotes or both, depending on which they are true for.

DNA is

packaged into chromosomes

DNA is the genet

ic mater

ial

Organisms have

a different sequence of bases along the

DNA

The DNA

is double

-strand

ed

DNA is

the genetic

material

DNA usually

found as a

double-stranded circular molecule

Plasmids can be

present

DNA not

found within

a nucle

us

DNA packaged into a set of linear

chromosomes

Mitrochondrial DNA

located within

the cytoplas

m

Chloroplast DNA

in some

species

Genetic material contained within

the nucleus

(b) DNA can direct its own replication (i) Replication of DNA Prior to cell division, DNA polymerase replicates a DNA strand precisely using DNA nucleotides.

DNA REPLICATIONAIM: DNA replication in the nucleus prior to cell division (INTERPHASE)

EQUIPMENT:1. DNA template2. Supply of each type of DNA nucleotide (Bases A,T,G,C)3. Energy supplied by triosphosphate molecules such as ATP.4. Appropriate enzymes-DNA polymerase (enzyme to join new nucleotides to the DNA strand) 5. Forward and reverse primers to start replication.

1. Template DNA unzips: Hydrogen bonds between base pairs break. The 2 anti-parallel strands are COMPLIMENTARY as nucleotides on one strand can be used as a template for the other.

Template – Nucleotide binding is as follows:Adenine----Thymine Cytosine---Guanine

2. PRIMERS: bind to the origin of replication to permit the DNA polymerase to bind to the DNA. Free nucleotides position themselves so that their bases can HYDROGEN BOND with the complimentary bases on each of the nucleotide strands [A-T] [C-G]. ATP supplies the energy for bond formation.

3. Free DNA nucleotides now in position, link up through sugar phosphate groups by forming DEOXIRIBOSE SUGAR PHOSPHARE BONDS. DNA polymerase catalyses addition of complementary nucleotides to the deoxyribose (3') end of the leading DNA strand. This occurs at several locations on a DNA molecule to speed up the process.

4. Problem: DNA polymerase only adds nucleotides in one direction 3’ to 5’

Solution: One strand (leading strand) is replicated continuously. The other strand (lagging strand) winds round twice in the DNA polymerase so that a short section can be read in the correct 3’ to 5’ direction. This is repeated so that the lagging strand is replicated in Okasaki fragments.

5. Fragments of DNA are joined together by ligase.

6. Once the whole length of the DNA has been replicated 2 DNA molecules are formed, which recoil into double helices identical to each other and the original.

This is the semi-conservative model of DNA replication.

Only DNA has all the requirements for genetic material essential to the continuation of life.1. Very stable: Coded instructions remain intact.from one generation to the next.2. Replicate: Can provide new copies of the genetic instructions for each new cell.3. Controls cells activities by controlling protein synthesis: DNA is aided by another nucleic acid

called RNA.

QUESTIONS1. What shape is the DNA molecule?2. What type of bonding holds two DNA strands together?3. Which two components make up a DNA molecule?4. Name the nitrogenous base that pairs with cytosine.5. Name the nitrogenous base that pairs with thymine.6. Which is the stronger bond: one between complementary bases A and T, or one between G

and C?

Semi-conservative replicationThe mechanism of DNA replication is said to be semi-conservative.That is, after replication, each of the two resulting DNA molecules is composed of one original (or conserved) strand and one new strand (middle diagram below).

This hypothesis, put forward by Watson and Crick, was proved experimentally by Meselson and Stahl in the late 1950s.

METHOD:1. Their experiment involved growing a culture of the bacterium Escherichia coli in a growth

medium containing heavy nitrogen (15N). 2. As the bacteria grew, they incorporated the heavy nitrogen into their nitrogenous bases. 3. The bacteria were subsequently inoculated into growth media containing light nitrogen

(14N) and 3 DNA classes were subsequently extracted after changing to the light medium. 4. The three classes of DNA were:

a. parental DNA. b._ first replication DNA; c. second replication DNA.

RESULTS:The results of the experiment showed that parental DNA grown in heavy medium was ’heavier’ than when grown in light medium. First generation growth showed that the DNA was all of medium density. Lastly, the second generation showed DNA of both medium and light intensities. Second generation growth supported the semi-conservative model of DNA replication since there were two bands of growth (one with both conserved and new DNA, and a band of light DNA).

Essay questionGive an account of the structure and replication of DNA.

REMEMBER:_ DNA is found on chromosomes inside the nucleus of the cell.

_ Chromosomal DNA is divided up into regions containing genes._ DNA consists of two strands twisted into a double helix._ DNA strands are made of nucleotides joined together. There are four types of nucleotides._ Each nucleotide consists of deoxyribose sugar, a phosphate group and one of four types of …organic base (adenine, guanine, cytosine or thymine)._ Base pairing rules - A always pairs with T and G always pairs with C.Polymerase Chain Reaction: The technique to produce DNA in vitroPCR is a technique that enables specific sections of DNA to be amplified (replicated) in vitro, producing millions of copies from a DNA template. PCR was developed by Kary Mullis in the mid 1980s, revolutionising molecular biology. He received the Nobel Prize for chemistry for its conception in 1993. Mullis developed the technique manually, and it can still be carried out using water baths. However, the technique is now fully automated in laboratories, using thermal cyclers no bigger than a bread machine.

The technique manipulates the cell’s natural mechanism for replication by using DNA polymerase and the following steps:

High energy bond like ATPPreparation of PCR mix which contains:

Dideoxynucleotides: (This is each base type A, T, G, C with their own phosphates added using high energy bonds. Like ATP this enables formation of the new DNA strand as energy for attachment of nucleotides is released when phosphates are removed)

Mg2+, which is a polymerase cofactor. Buffer to keep the pH stable. Template DNA (Blood, hair root, bacterial colony) Forward and reverse primers Taq DNA polymerase

The sample is placed in a thermocycler where the temperature cycles through 3 settings to replicate DNA.1. Sample DNA is denatured by heating to

give two polynucleotide chains.2. Sequence-specific primers, which are small

sequences of single-stranded DNA, typically of 8–15 base pairs in length, anneal to the DNA flanking the section of interest. One primer anneals to one strand (forward primer),another to the other DNA strand (reverse primer).

3. Polymerase begins to replicate the DNA sectionof interest using the primers as a starter sequence.

4. The mixture now contains the original template plus the newly amplified sections.

5. The cycle begins again using original & copied DNA as templates. As the reaction is exponential, millions of copies are produced in about 3 hours.

Figure 6: The polymerase chain reaction.

Applications of PCRPCR is used widely, for example:1. DNA profiling/fingerprinting:

PCR is used to rapidly identify individuals. Specific regions of DNA known to vary between individuals are amplified using fluorescently labelled primers and then analysed using capillary gel electrophoresis. Profiling is not only used in forensics but also in plant variety identification, paternity testing and evolutionary biology.

2. Disease diagnosis: DNA sequences that are known to indicate certain genetic disorders or diseases are amplified using PCR for the purposes of diagnosis.

3. Archaeological analysis: Ancient DNA, degraded over the years, can be amplified and used in archaeological, paleontological and evolutionary research.

4. Population studies: Analysis of human or other species’ population genetics can be rapidly performed using PCR analysis.

5. Sequencing: DNA sequence analysis previously took place following lengthy cloning experiments, which have now been replaced by PCR.

QUESTIONS:Q1: DNA is present in which part of the cell?

a) Cytoplasmb) Nucleusc) Cytoplasm and nucleus

Q2: DNA is made of which of the following?a) Nucleotidesb) Amino acidsc) Fatty acidsd) Glucose molecules

Q3: What is the name of the monomers that make proteins?a) Nucleotidesb) Amino acidsc) Fatty acidsd) Glucose molecules

For further information about PCR refer to the following website:http://www.dnalc.org/resources/animations/pcr.html

A song about PCR can be seen here:http://www.youtube.com/watch?v=x5yPkxCLads

And there is a PCR rap here: http://www.youtube.com/watch?v=oCRJ4r0RDC4&feature=related

RNA Structure and Protein Synthesis

Gene Chromosomes mRNA Polypeptide(s) Protein (sequence of 3 bases)This Topic explores how information flows from gene to protein.

RNA structureRibose Nucleic Acid (RNA) consists of nucleotides with the generalised structure:

The RNA nucleotides are joined to form a single strand. RNA nucleotides are joined by a bond between the sugar and the phosphate of the next molecule.

Comparison of DNA and RNANucleic acid DNA RNA . Sugar Deoxyribose Ribose.Bases A (adenine), G (guanine), A (adenine), G (guanine), T (Thymine), C (cytosine) C (cytosine), U (uracil)._ RNA has nitrogenous base uracil rather than thymine.

RNA is a type of nucleic acid called ribonucleic acid:

Structure Double stranded Single stranded.Location Nucleus Nucleus and cytoplasm

FUNCTION OF RNA: Ribonucleic acid (RNA) provides a bridge between DNA and protein synthesis.

1. messenger RNA , or mRNA . 2. transfer RNA , or tRNA . Formed: In the nucleus. Found: In the cytoplasm.Function: It rewrites the sequence of bases Function: The tRNA picks up specific amino acids from ………………of a section of DNA in a process cytoplasm and brings them into position on ……………..called transcription. the surface of a ribosome where they can bemRNA carries the code for building a specific joined together in specific order to make a specific protein from the nucleus to the ribosomes protein. This process is called TRANSLATION in the cytoplasm. It acts as a messenger.Questions 1. Name the three components of an RNA nucleotide.

2. Which base is found in RNA but not DNA?

3. Is RNA single-stranded or double-stranded?

Protein synthesis

Process whereby DNA encodes for the production of amino acids and proteins.

This process can be divided into two parts:

1. Transcription (Takes place in the nucleus)

the gene coding for the protein required untwists then unzips, the H-bonds between the strands break

free RNA nucleotides form complementary base pairs with one strand of DNA bases

weak hydrogen bonds form between base pairs sugar phosphate bonds form between RNA

nucleotides mRNA strand is synthesized mRNA peels off the DNA and moves out of the

nucleus into the cytoplasm

2. Translation (Takes place in the cytoplasm)

the ribosomes are the sites of protein synthesis

the mRNA strand attaches to a ribosome tRNA molecules transport specific amino acids

to the ribosome each mRNA codon codes for a specific amino

acid

the anti-codons and codons match up and form complementary base pairs peptide bonds form between the adjacent amino acids to form the polypeptide (protein)

tRNA is reused and collects another specific amino acid. Once the protein has been synthesised mRNA may move to another ribosome to make a further protein or it can be broken down into free nucleotides to be reused.

Aim: To copy part of one strand of DNA into a single stranded mRNA molecule.Apparatus:DNA template RNA nucleotides ATP RNA polymerase

Transcription of DNA into mRNATranscription is the first step in protein synthesis. 1. . Before protein synthesis begins, the corresponding RNA molecule is produced by RNA transcription. 2. The 2. DNA double helix unwinds andUnzips in a short section,exposing a single template strand of DNA.

…

3. One strand of the DNA double helix is used as a template by the RNA polymerase to synthesize a ….messenger RNA (mRNA). Free RNA nucleotides in the nucleus bind to complementary DNA nucleotides …using the base pair rules DNA nucleotide RNA nucleotide A pairs with U T pairs with A G pairs with C C pairs with G

4. The RNA molecule produced during transcription of DNA is called PRE-messenger RNA(pre-mRNA) and is a faithful transcript of the DNA template.

5. Pre-mRNA migrates from the nucleus to the cytoplasm.

RNA SPLICING

During this step, mRNA goes through different types of maturation including one called splicing when the non-coding sequences (introns) are eliminated.

6. The coding regions (exons) are ligated together to form mRNA. The coding mRNA sequence can be described as a unit of three nucleotides called a codon.

Translation of mRNA into proteinOnce the DNA in a gene has been transcribed into mRNA, translation can take place.

Aim: To complete translation of the codons on mRNA into amino acids linked by peptide bonds (protein) Translation of mRNA into protein.

Location: On ribosomes in the cytoplasm Apparatus:Transfer RNA (tRNA) Messenger RNA (mRNA) Ribosomal RNA (rRNA)

Introns: Intervening sequences which are not part of the protein being produced.

Introns may be:1.Promoters for RNA transcription2.They may be part of another protein3.

Pre-mRNA with introns removed is called mRNA.

The mRNA molecules pass through the nuclear pores to the cytoplasm the site protein synthesis and the processes of:

1. Translation2. Post-translational

modification

tRNA folds due to base pairing to forma triplet anticodon site and anattachment site for a specific amino acid.

TRANSLATION-Translation of mRNA into a polypeptide by tRNA at the ribosome.

tRNA folds due to base pairing to form a triplet anticodon site and an attachment site for a specific amino acid. Triplet codons on mRNA and anticodons translate the genetic code into a sequence of amino acids. Start and stop codons exist. Codon recognition of incoming tRNA, peptide bond formation and exit of tRNA from the ribosome as polypeptide is formed.

Translation of mRNA by the ribosome occurs in three stages: initiation, elongation, and termination.

Initiation1. The small ribosomal subunit binds to the start of the mRNA sequence. 2. Then a transfer RNA (tRNA) molecule carrying the amino acid methionine binds to what is called the

start codon of the mRNA sequence. 3. The start codon in all mRNA molecules has the sequence AUG and codes for methionine. 4. the large ribosomal subunit binds to form the complete initiation complex.

Elongation stage1. The ribosome continues to translate each mRNA codon in turn. 2. The tRNA molecule carrying the complementary anticodon binds briefly to the mRNA codon. 3. The amino acid attached to the tRNA is then added to the polypeptide chain being synthesised.4. Each corresponding amino acid is added to the growing chain and linked via a bond called a

peptide bond. Elongation continues until all of the mRNA codons are read.

Termination,1. When the ribosome reaches a stop codon (UAA, UAG, and UGA). 2. Since there are no tRNA molecules that can recognize these codons, the ribosome recognizes that

translation is complete. The new protein is then released, and the translation complex comes apart. 3. After the amino acid has been added to a polypeptide chain during translation, the tRNA is free to

pick up another amino acid in the cytoplasm.

As a mRNA molecule passes through a ………..ribosome each codon is translated into an

an amino acid.

One specific amino acid can correspond to … more than one codon.

The genetic code is said to be.degenerate.

The table shows how the genetic code indicates which amino acid corresponds toeach mRNA codon.

Notice that more than one mRNA codon codes for each amino acid. Some tRNA molecules have anticodons that recognise two or more different codons. This relaxation of the base pairing rules is called wobble. This explains why the mRNA codons GUU, GUC, GUA and GUG, for example, all code for the same amino acid, valine. tRNA molecules pick up the appropriate amino acids in the cytoplasm of the cell.

SUMMARY OF TRANSCRIPTION AND TRANSLATION

RNA and protein synthesis exam skillsQuestions on this topic often require you to demonstrate an ability to deal with the complementary base pairings between DNA, mRNA and tRNA. You need to understand the progression working forward from DNA to mRNA then to tRNA and to the associated amino acid may be provided in a table. Other questions may reverse this order of events. To make the task more manageable draw a diagram showing the base pairings to allow you to work forward or backwards as required.

Example 1Part of a DNA strand has the base order A T C G T T C A G. You are asked to identify the anti-codons associated with this strand. Remember the sequence of events in the stages of RNA protein synthesis:

transcription of mRNA from DNA tRNA matches the mRNA (anti-codons match the codons)

You should then work forward identifying the triplet codes as follows (make sure you substitute uracil (U) for thymine (T) when dealing with the RNA molecules):

Triplet codes in DNA, mRNA, and tRNADNA strand:ATC GTT CAGmRNA :UAGCAA GUCtRNA :AUCGUUCAG

Processing and secretion of proteins

Proteins made in ribosomes that are attached to the endoplasmic reticulum are secretedout of the cell. This process is illustrated in an on-line example that also gives adescription of the stages involved in protein processing and secretion.

After translation, the protein passes into the channels of the rough endoplasmic reticulum (ER) for transportation. The protein is then passed from the rough ER to the Golgi apparatus inside tiny fluid-filled sacs, called vesicles. The Golgi apparatus is a system of membranes, which are responsible for the modification, processing, and packaging of the proteins. The protein may have a carbohydrate added, to form a glycoprotein. The Golgi apparatus packages the protein in a secretory vesicle, which fuses with the cell membrane and releases the protein from the cell.

Questions:1: Where are proteins initially processed?a) Golgi apparatusb) Ribosomesc) Mitochondriad) Endoplasmic reticulum

2: What name is given to proteins that contain a carbohydrate component?a) Amino acidsb) Insulinc) Glycoproteinsd) Polypeptides

3: What name is given to the process of secretion of intracellular products by the cell?a) Endocytosisb) Exocytosisc) Lysisd) Phagocytosis

Essay: protein synthesisGive an account of protein synthesis under the following headings:1. transcription; (4)2. translation. (6)

Functional Variety of Proteins

Proteins are essential in all biological systems.

Protein functions:

1. Structure 2. Metabolism 3. Energy (only in starvation)

The structure of proteins are related to their function:Structural proteins are fibrous Metabolic proteins are globular

Proteins are made of long chains of amino acids.There are 20 different amino acids.They are joined together by peptide bonds.

Proteins:are polymers, made up of amino acids joined together; the type and order of amino acids determines protein structure;_ interactions between amino acids in polypeptide chains defines the structure and function of the protein.

MAKING PROTEINS FROM POLYPEPTIDESThe same gene can be used to make several different proteins by:1. Alternative RNA splicing 2. Post-translational modification

1. Alternative RNA splicing (AS BEFORE)The primary transcript is separated into exons and introns and the exons are spliced together to make the mature transcript.

Under certain conditions alternative segments of RNA may be treated as exons and introns.

In other words one gene can produce several different mature mRNA transcripts and therefore, several different proteins.

2. Post-translational modification

Once translation is completed, proteins can be modified by:(a) Cleavage(b) Addition of other molecules

(a) Cleavage

A single poly-peptide chain can be cleaved (cut) by enzymes to make it active.

Insulin is an example of the protein modified in this way.

A central section of the “pro-insulin” molecule is removed to make insulin.

(b) Addition of other moleculesCarbohydrates and phosphate groups can be added to proteins.Mucus is a glycoprotein made by the addition of a protein and a carbohydrate.

Some regulatory proteins need phosphate groups added to them to make them active.

e.g. p53 is a regulator involved in DNA repair. Normally it is inactive. When phosphate is added it becomes active.

Proteins are organised at four different structural levels:

_ Primary (sequence of amino acids linked by PEPTIDE BONDS);

_ Secondary (weak HYDROGEN bonds between amino acids cause the polypeptide chain to fold);Result: alpha-Helices or Beta-barrels

_

Tertiary (strong bonds form between amino acids causing more folding to take place);_

_ Sulphur atoms, which are found in some amino acids, can also lead to theformation of links within a polypeptide, or between different polypeptides.

_ Fibrous proteins, Globular proteins, Conjugated proteinse.g. collagen, e.g. enzymes, e.g. Glycoproteins are formed from polypeptide are highly folded, chemically modified or chains linked in parallel coiled structure contain a non-protein part

_ Quaternary (two or more polypeptide chains often associate together to form the final protein structure).

Haemoglobin is an example of a protein with a quaternary structure. It is found in red blood cells and is responsible for carrying oxygen around the body. It is composed of four chains of the protein globin, with two each of alpha- and beta-globin.. Protein structure is affected by various factors including: 1. pH; 2. _ temperature Any shift from optimal conditions reduces the ability of a protein to function properly. A major shift causes it to denaturation and all function is lost.

Globular proteins include the following:_ enzymes;

_ many membrane proteins_ some hormones;_ antibodies.

Structural (fibrous) proteins include the following:CollagenElastin

QUESTIONS

Q1: Which of the following statements describes antibodies?a) They are part of the cell membrane.b) They cut, assemble or digest other molecules.c) They make holes in the cell membrane.d) They recognise molecules of invading organisms.

Q2: Proteins are composed of which of the following?a) Nucleotidesb) Glucose moleculesc) Glycerol moleculesd) Amino acids

Q3: A polypeptide chain is held together by which of the following?a) Weak hydrogen bondsb) Weak peptide bondsc) Strong hydrogen bondsd) Strong peptide bonds

Q4: The description "The peptide is coiled around an imaginary cylinder and stabilisedby hydrogen bonds" is true for which of the following?a) Alpha helixb) Fibrous proteinc) Globular proteind) Structural protein

Q5: Glycoproteins are composed of proteins containing which of the following?a) Carbohydrateb) Nucleotidesc) A non-protein chemicald) Fatty acids