Lecture 17 Transcription translation Flashcards
What are major phenotypic differences the result of?
Differences in specific proteins
What are the two major steps that express a gene?
Transcription and translation
What is transcription?
Information of a DNA sequence is copied into the corresponding information in an RNA sequence
What does translation do?
Convert RNA sequences into an amino acid sequence of a polypeptide
What is the purpose of RNA?
To be an intermediary between DNA and proteins
How does ribonucleic acid differ from DNA?
RNA is only one polynucleotide strand
RNA has ribose, rather than deoxyribose
RNA has uracil instead of thymine
How is uracil different to thymine?
It lacks a methyl group
What is the central dogma?
DNA codes for the production of RNA
RNA codes for the production of protein
Protein does not code for RNA or DNA
What theory did Crick propose to explain how information gets from the nucleus to the cytoplasm?
Messenger hypothesis
What is the messenger hypothesis?
RNA forms as a complementary copy of DNA of a particular gene
This messenger RNA (mRNA) travels from the nucleus o the cytoplasm
What did Crick propose to explain how a DNA sequence gets transformed into the specific amino acid sequence of a polypeptide?
The adapter hypothesis
What is the adapter hypothesis?
There must be an adapter molecule that can bind a specific amino acid sequence and recognize a sequence of nucleotides
What were found to be adapter molecules?
Transfer RNA (tRNA)
Why is it said that tRNA translates DNA?
Because they recognize the genetic message of mRNA and simultaneously carry specific amino acids
What happens during translation?
tRNA carrying bound amino acids line up on the mRNA sequence so the amino acids are in the correct sequence
How has the adapter hypothesis been confirmed?
Actual observation
What exceptions are there to the central dogma?
RNA viruses
Give some examples of viruses that have RNA genetic material.
Tobacco mosaic virus, influenza virus, poliovirus
How do RNA viruses replicate?
They make a complementary RNA strand to their genome which is used to make copies of the viral genome by transcription
What is synthesis of DNA from RNA called?
Reverse transcription
Name a retrovirus.
HIV
What type of genetic information does HIV have?
RNA
How does HIV replicate after infecting a host cell?
By making a DNA copy of their genome and using it to make RNA to use as a template and as mRNA to produce viral proteins
What enzyme is used in reverse transcription to synthesize DNA from RNA?
Reverse transcriptase
What components are needed for transcription?
- DNA template
- Ribonucleoside triphosphates (ATP, GTP, CTP, UTP) (substrates)
- RNA polymerase enzyme
What name is given to strand that is transcribed during transcription?
The template strand
What is the name given to the complementary strand that is not transcribed?
Non-template strand
What is synthesized during transcription?
mRNA, tRNA ribosomal RNA (rRNA)
What does RNA polymerase do?
Catalyses the addition of nucleotides in the 5’ to 3’ direction, processive
How many RNA polymerases are there in bacteria?
one
How many RNA polymerases are there in eukaryotes?
Three
What is the common structure of RNA polymerases?
Crab claw structure
What is the first step in RNA polymerase catalysis?
The enzyme recognizes certain bases within the DNA double helix and binds to them
What does RNA polymerase do after binding to certain bases on the DNA?
The ‘pincers’ close, keeping DNA in a double strand form called a closed complex
What happens once RNA polymerase has bound to DNA with a closed complex?
A conformational change in the RNA polymerase occurs, denaturing a short (10 base pairs) stretch of DNA and forming an open complex.
What happens after an open complex has formed?
Unpaired bases within DNA are available to pair with ribonucleotides, RNA synthesis begins
In what direction does the RNA polymerase read the DNA template strand?
from 3’ to 5’
How is RNA polymerases different from DNA polymerases during initiation?
Does not require a primer.
What three processes can transcription be divided into?
- Initiation
- Elongation
- Termination
What step begins transcription and what is required?
Initiation
A promoter
What is a promoter?
A sequence of DNA to which RNA polymerases bind very tightly
What do promoter sequences tell RNA polymerases?
- Where to start transcription
- Which strand of DNA to transcribe
- The direction to take from the start
How many promoters are there?
At least one for each gene
What part of the promoter is where transcription begins?
The initiation site
When does elongation start?
Once the RNA polymerase is bound to the promoter
How are DNA polymerases different to RNA polymerases during elongation?
RNA polymerases do not proof-read their work
Why are RNA polymerase errors not as serious as DNA polymerase errors?
Many copies of RNA are made, and they have a relatively short life span
(Errors are not as harmful)
What is termination controlled by?
Specific sequences of bases
How is termination of transcription different in prokaryotes compared to eukaryotes?
Prokaryotes- translation can start before transcription is finished
Eukaryotes- spatial separation, pre-mRNA
What is a codon?
Three nucleotides in a messenger RNA that direct the placement of a particular amino acid into a polypeptide chain
What is a start codon?
The initiation signal for translation
What is a stop codon?
Termination signal for translation
What happens when translation machinery reaches one of the stop codons?
Translation stops, the polypeptide is released from the translation complex.
What is the codon for a start codon?
AUG
What are the three stop codons?
UAA
UAG
UGA
What is the name given to genetic code to describe how there is more than one codon for each amino acid?
Redundant
What two amino acids are represented by just one codon each?
Tryptophan
Methionine
What other quality describes the genetic code?
It is not ambigous
What does it mean that genetic code is not ambigous?
A codon can only code for one amino acid
What does it mean that the genetic code is (nearly) universal?
In almost every species, codons that specify amino acids are the same
What are does the universal nature of the genetic code imply?
That the code is ancient and has been maintained throughout the evolution of living organisms
Where are some exceptions to the universal nature of genetic code be seen in plants?
Within mitochondria of chloroplasts the code differs
Other than plants, where can exceptions to the universal nature of the genetic code be seen?
In one group of protists, UAA and UAG code for glutamine rather than functioning as stop codons
What 2 key events during translation must take place to ensure that a protein made is the one specified by mRNA?
- tRNA must read mRNA codons correctly
- tRNA must deliver the amino acids that correspond to the mRNA codons it has read
What happens after tRNA’s have delivered the appropriate amino acids?
The ribosome catalyzes the formation of peptide bonds between amino acids
What are the three functions of the tRNA molecule?
- Carries (is charged with) an amino acid
- Associates with the mRNA molecules
- Interacts with ribosomes
How many nucleotides does a tRNA molecule have?
75-80
How is the conformation of a tRNA molecule maintained?
Complementary base pairing (hydrogen bonding) within its own sequence
What is at the 3’ end of every tRNA molecule?
Its amino acid attachment site
How do amino acids attach to the 3’ end of the tRNA molecule?
By binding covalently
What is at the mid point of the tRNA sequence?
A group of three bases called the anticodon
What happens at the anticodon?
The site of complementary base pairing via hydrogen bonding to the mRNA.
Why aren’t there 61 different tRNA species?
Specificity for the base at the 5’ end of the anticodon is not always observed- this is called wobble
Give an example of wobble.
Alanine codons GCA, GCC and GCU are recognized by the same tRNA
How is the charging of each tRNA with its correct amino acid achieved?
By a family of activating enzymes known as amino-acyl-tRNA synthetases
What are each activating enzymes specific to?
Corresponding tRNA
How many parts make up the active site of amino-acy-tRNA synthetases?
3 parts that recognize three smaller molecules
What smaller molecules are recognized by amino-acy-tRNA synthetases?
A specific amino acid
ATP
A specific tRNA
Why is the process of tRNA charging known as the second genetic code?
Because the activating enzymes are highly specific
What is the first step in tRNA charging?
The enzyme activates the amino acid, catalyzing a reaction with ATP for form high energy AMP-amino acid and a pyrophosphate ion
What is the second step in tRNA charging?
The enzyme then catalyzes a reaction of the activated amino acid with the correct tRNA
What happens after the enzyme has catalyzed a reaction of the activated amino acid the the correct tRNA?
The charged tRNA delivers the appropriate amino acid to join the elongating polypeptide product
Why is the error rate for the activation enzyme’s recognition of tRNA very low?
Because the tRNA is large with a complex structure
Where does the energy for the synthesis of the peptide bond for joining adjacent amino acids come from?
The energy rich bond formed between the 3’ end of the tRNA with an amino acid
Where does translation take place?
The ribosome
What subunits is a ribosome made up of?
A small subunit (40S)
A large subunit (60S)
What is the function of the ribosomes?
To hold mRNA and tRNA for polypeptide synthesis
What are the different molecules that make up the large subunit in eukaryotes?
Ribosomal RNA (rRNA) As well as 45 protein molecules arranged in a precise pattern
What sites make up the large subunit?
The A site (amino acid)
The P site (polypeptide)
The E site (exit)
What is the small subunit made of in ribosomes?
One rRNA molecule and 33 different protein molecules
How do ribosomes exist when not active?
As separate molecules
How do ribosomes differ among prokaryotes and eukaryotes?
Smaller in prokaryotes
Different rRNA’s
How are the proteins and rRNA’s of ribosomal subunits held together?
Ionic and hydrophobic forces (not covalent bonds)
What happens at the A site?
Charged tRNA anticodon binds to the mRNA codon
What happens at the P site?
tRNA adds its amino acid to the growing polypeptide chain
What happens at the E site?
tRNA has given up its amino acid, resides before being released and going back to the cytosol to pick up another amino acid.
What is an important role of the ribosome?
To make sure that the mRNA-tRNA interactions are accurate
How does the small ribosomal subunit ensure the mRNA-tRNA interactions are accurate?
If hydrogen bonds have not formed between all base pairs, the tRNA must be wrong, the tRNA is ejected
What three steps does translation take place in?
Initiation, elongation, termination
How does the initiation of translation of mRNA begin?
The formation of an initiation complex
What is an initiation complex?
A charged tRNA bearing the 1st amino acid and a small ribosomal subunit both bound to the mRNA
What does the rRNA of the small ribosomal subunit first bind to?
A complementary ribosomes binding site on the mRNA
What is the sire that rRNA first binds to known as?
The shine-Dalgarno sequence
Where abouts is the Shine-Dalgarno sequence compared to the start codon?
Up stream- towards the 5’ end
What event completes the formation of the initiation complex?
The anticodon of a methionine charged tRNA binds to the start codon by complementary base pairing
Why do not all proteins have methionine as their N-terminal amino acid?
Because the initiator methinonine can be removed by an enzyme after translation
What happens after the methionine-charged tRNA has bound to the RNA?
The large subunit of the ribosome joins the the complex
The charged tRNA now lies in the P site
What are the mRNA, two ribosomal subunits and methionine-charged DNA known as?
Initiation factors
What two reactions does the large subunit of the ribosome catalyze during elongation?
- Breaks the bonds between tRNA in the P site and its amino acid
- Catalyzes the formation of a peptide bond between that amino acid and the one attached to the tRNA in the A site
What is the large subunit said to have because it performs two actions?
Peptidyl transferase activity
What assists the elongation steps of translation?
Elongation factors
How does termination begin?
A stop codon enters the A site
What do stop codons bind?
A release factor, which allows hydrolysis of the bond between the polypeptide chain and the tRNA of the P site
The formation of what increases the rate of protein synthesis?
Polysomes
How can several ribosomes work together simultaneously?
As soon as the Shine-Dalgarno sequence is far enough from the ribosome, a second initiation complex can form, and then a third…etc.
What is the assemblage consisting of a strand of mRNA and beadlike ribosomes called?
polyribosome, or polysome
What are two posttranslaional aspects of protein synthesis?
Transport and modification
How are proteins directed to their cellular destinations?
Signal sequences
What happens to the polypeptide chain as it emerges from the ribosome?
It folds into a 3-D shape determined by amino acid sequence
What is a signal sequence?
An amino acid sequence that indicates where in the cell the polypeptide belongs.
What two instructions can a polypeptide chain give to a ribosome during translation?
- Finish translation and be released to an organelle
- Stop translation, go to the endoplasmic reticulum and finish synthesis there
What happens to proteins that instruct the ribosome to finish translation and be released to an organlle?
They are sent to the nucleus, mitochondria, plastids, peroxisomes, depending on the address.
What happens if a polypeptide lacks instructions?
They remain in the cytosol
What happens to proteins that give instructions to stop translation, go to the endoplasmic reticulum and finish synthesis there?
After protein synthesis is complete, these proteins are retained in the ER and sent to the golgi apparatus, where they may be sent to lysosomes, plasma membrane, or outside of the cell
What happens to proteins lacking instructions in the endoplasmic reticulum?
They are packaged into vesicles and secreted from the cell
Where are localization sequences found on a protein?
At either the N or C terminus, or interior of chain
Give an example of proteins that would go to the nucleus?
Histones
Give an example of proteins that would go to the mitochondria.
citric acid enzymes
How do signal sequences work?
They have a conformation that allows them to bind to specific receptor proteins called docking protein
Where are docking proteins?
On the outer membrane of the appropriate organelle
What happens once a protein has bound to a docking protein?
The receptor forms a channel in the membrane for the protein to pass through
How does the protein pass through the receptor channel?
It is unfolded by a chaperonin then refolds on the other side.
What is needed for a protein to be seen to the ER and then to the golgi?
A specific hydrophobic sequence of 15-30 amino acids at the N terminus
What happens to the sequence needed to send a protein to the ER before translation is finished?
The sequence signal binds to a signal recognition particle composed of protein and RNA
What does binding of a protein’s sequence signal to a signal recognition particle do?
Blocks further protein synthesis until the ribosome attaches to a specific receptor protein in the membrane of the rough ER
What happens once the ribosome attaches to a specific receptor protein in the membrane of the rough ER?
The receptor protein is converted into a channel
What are modifications of proteins for?
Essential to the final function of the protein
What are three types of protein modification?
Proteolysis
Glycosylation
Phosphorylation
What is proteolysis?
Cutting of a polypeptide chain
Give an example of proteolysis.
Cleavage of the signal sequence form the growing polypeptide chain in the ER
Why is it important to remove the signal sequence from a growing polypeptide chain in the ER?
The protein might move back out
What are polyproteins?
Long polypeptides cut into final products by proteazes
What is glycosylation?
The addition of sugars to proteins to form glycoproteins
Why is glycosylation important?
Adding sugars is important for targeting and recognition
Why is proteolysis important?
Cleaving polypeptides allows fragments to fold into different shapes
What is phosphorylation?
The addition of phosphate groups to proteins, catalyzed by protein kinases.
Why is phosphorylation important?
Added phosphate groups alter the shape of the protein.
What are the two types of mutations in multicellular organisms?
Somatic mutations
Germ line mutations
What are conditional mutants?
Mutations that cause their phenotype only under certain restrictive conditions
They are not detectable under permissive conditions
What are many conditional mutants sensitive to?
Temperature
Why are many conditional mutants sensitive to temperature?
The mutant allele may code for an enzyme with an unstable tertiary structure that is altered at restrictive temperatures
What are mutations?
Alterations in the nucleotide sequence of DNA at the molecular level.
What two categories can mutations be divided into?
- Point mutations
- Chromosomal mutations
What are chromosomal mutations?
Changes in position and orientation of DNA without removing it, or by losing or duplicating segments of DNA
What are point mutations?
Mutations of single base pairs limited to single genes
How do point mutations occur?
Errors in DNA replication,
Environmental mutagens
What are silent mutations?
Base substitutions that result in no change in amino acids because of the redundancy of the genetic code
What is the result of silent mutations?
Genetic diversity not expressed as phenotypic differences
What are missense mutations?
Base substitutions that change the genetic message such that an amino acid substitutes for another
Give an example of a missense mutation.
Sickle allele for human beta-globin
What is sickle-cell disease?
A disease from a defect in haemoglobin
What causes the sickle-cell disease?
The sickle cell allele of the gene that codes for beta-globin is different by one base, codes for a different polypeptide
What are nonsense mutations?
One base is substituted for another which causes a stop codon to form in the mRNA product
What is the result of a nonsense mutation?
A shortened protein that is usually not functional
What is a frameshift mutation?
Single base pairs are inserted or deleted from DNA, interferes with decoding of genetic code by throwing it out of register
What do frameshift mutations lead to?
The production of non-functional proteins
What types of chromosomal mutations can occur?
- Deletions
- Duplications
- Inversions
- Translocations
What is the 5’ cap?
Methylated guanosine triphosphate is added to the end of the pre-mRNA transcript
What are the 2 functions of the 5’ cap?
- Facilitates transport and binding to ribosome
- Protects the transcript from ribonucleases
What is added to the 3’ end of the pre-mRNA molecule?
A poly A tail
What is a poly A tail?
AAUAAA signals for one enzyme to cut the pre-mRNA and another enzyme to add 100-300 adenine nucleotides
What is the purpose of the poly A tail?
Assist export of the transcript from the nucleus
Ensures mRNA stability
What are introns and exons?
Exons code for polypeptide product
How are introns removed?
Splicing
How does splicing work?
snRNPs bind to intron and exon boundaries, then come together with other proteins to form the spliceosome
Introns are removed, exons are spliced together.
What happens after the pre-mRNA transcript has been processed into the mature mRNA?
the cap binding complex binds to the 5’ cap.
What is the purpose of the cap binding complex?
This complex is recognised by a receptor in the pores
of the nuclear membrane.
The mature transcript can then be transported out of
the nucleus and into the cytoplasm for translation.
