BIO 1A03 McMaster University Test #2 Review

Translation

Cellular components are able to read the genetic message in mRNA and translate the message into specific primary amino acid sequence of a protein

How does tRNA structure allow for SPECIFICTY in translation?

1. Plays critical role in ENABLING the translation of the information
2. Each type of tRNA molecule is not identical and can translate a specific codon into a specific amino acid

What is the structure of tRNA?

- single RNA strand (70-90 nucleotide length)
- four double helical segments and three characteristic loops

How are the four double helical segments and three loops in tRNA molecules formed?

Formed due to the large degree of complementarity along stretches of tRNA which results in many stretches of hydrogen bonding between complementary bases.

What are anticodons? In what direction are they conventionally written?

Three-base sequence in a transfer RNA molecule base that pairs with a complementary codon in mRNA

They are conventionally written in the 3' to 5' direction

What is at the 3' end on tRNA?

There is a protruding amino acid attachment site made up of CCA nucleotide sequence (terminal "A" is point of attachment for amino acid during tRNA molecule activation)

How is the activation of a tRNA molecule with a specific amino acid carried out?

Activation is carried out by a family of enzymes called aminoacyl tRNA synthetase

True or False; Each enzyme is specific to the type of tRNA and corresponding amino acid it will bind.

True - 20 aminoacyl tRNA synthetase

How do aminoacyl tRNA synthetases work?

The active site of the enzyme recognizes the anticodon end of the tRNA and the region of the amino acid attachment site.

Once bound to active site, enzymes catalyze covalent attachment of tRNA molecule to its amino acid (energy from ATP Hydrolysis)

Charged tRNA

able to deliver amino acids to growing polypeptide chain.

What does correct translation require (in terms of codon and anti-codon)?

Correct translation requires the correct PAIRING of tRNA anticodon with the appropriate mRNA codon

How does codon-anticodon pairing occur?

The 5' end of codon (mRNA) will bind with the 3' end of the anticodon (tRNA)

What is meant by the term wobble? What does it help to explain?

Greater flexibility for base pairing between third nucleotide of a codon and corresponding base of tRNA anticodon

Wobble helps explain the redundancy of genetic code

When does translation initiation in Eukaryotes occur?

In eukaryotes, it occurs when a translational initiation complex forms towards the 5' cap of the mRNA and then scans the mRNA until an AUG start codon is encountered

How does translation initiation in Prokaryotes occur?

Since there are no 5' caps, translation initiation complex will assemble at one or more ribosome binding sites called Shine-Dalgarno sequences (Located a few bases upstream the translation start codon)

What is the unique ability of prokaryotic translation? (think operons)

Translation can occur along multiple regions of prokaryotic mRNA sequence
- Allows prokaryotes to have specific open reading frames for more than one protein along mRNA

Eukaryotic translation initiation requires what components?

Two ribosomal subunits
mRNA
Charged tRNA
Initiation factors

Initiation factors

Bind to 5' cap of mRNA which allows for the recruitment of the small ribosomal subunit

Other initiation factors will bind to tRNA that is charged with methionine

The partially assembled small subunit and tRNA do what once initiated?

They will move along the mRNA in a 5' to 3' direction until AUG is encountered
- When this occurs, large ribosomal subunit is able to bind to the rest of the initiation complex using energy from GTP hydrolysis

What happens to initiation factors once translation is complete?

Initiation factors are released once translation is complete

How does translation start in the ribosome (not initiation)?

- Methionine located in peptidyl site of ribosome
- As the ribosome translates the mRNA, each subsequent charged tRNA enters or binds to aminoacyl (A) site

GTP-bound elongation factor

Each incoming charged tRNA is delivered in association with these

When the correct codon-anticodon pairing is made, GTP is hydrolyzed and aminoacyl end of tRNA is released from elongation factor

How are peptide bonds formed in translation?

Once tRNA binds mRNA, a conformational change occurs that is induced in ribosomal RNA that allows for peptidyl-transferase reaction to occur

The condensation reaction occurs as peptide bond and transfers amino acid to growing polypeptide chain onto tRNA in A-site

When another tRNA binds the site, what happens?

The E-site is ejected

How is translation terminated?

Once ribosome reaches stop codon, GTP-bound release factors will bind A-site and catalyze hydrolysis of bond between terminal amino acid in polypeptide and tRNA in P-site

Further GTP hydrolysis will also enable the dissociation of the translation complex

one gene-one enzyme hypothesis

the function of a gene is to dictate the production of a specific enzyme

- Based on the fact that Neurospora can grow well on minimal medium
- They must have some enzymes produced by a specific gene that convert simple substances into amino acids and vitamins necessary for growth

Neurospora are able to synthesize what?

Able to synthesize arginine through metabolic pathway

What did Srb and Horowitz demonstrate?

Each gene contains the information needed to make each enzyme

How did Srb and Horowitz test their hypothesis?

- Performed genetic screen of radiation treated neurospora to determine if there are specific enzymes produced in the metabolic buildup of arginine
- Radiation would lead to mutations so they conducted the genetic screen by raising colonies of radiation treated cells on a medium that was supplemented with nothing, ornithine, citrulline, or arginine added to medium

What was the result of Horowitz and Srb's experiment?

1. With supplemented arginine, fungus is able to undergo growth
2. Non-supplemented medium, fungus not able to grow
3. Orthinine and Citrulline only: Inhibition in growth

Identified 3 mutants: Arg 1 (grows in all), Arg 2 (Citrulline and arginine only), Arg 3 (arginine growth only)

What is the exception to the one-gene, one enzyme hypothesis?

- 20-25,000 protein encoding genes in human genome
- Provided evidence that more than one protein can possibly be produced from a single gene
- Alternative splicing + other mechanisms contribute to complexity of mRNA and therefore protein

The Human Proteome

The full number of proteins that are expressed by all the hereditary information in our DNA. (20-25,000 protein encoding genes)

The complexity of our proteome relative to our genome is attributed to what?

The great complexity is largely attributed to RNA processing and post translational modifications

True or False, the composition of proteome can change in response to various factors?

True - Occurs in organisms developmental stage and in response to internal and external signals

How does a cells ability to detect changes in environment benefit the cell?

Changes serve as stimuli that will result in important cellular responses

What happens in your body after a meal?

1. Stimulus can include an increase in Blood glucose levels (regulated by sensory responses in specialized islet cells of pancreas)
2. Will lead to a cascade of events that return blood glucose back to normal

What happens in the case of high blood glucose?

The pancreas will modulate the synthesis and secretion of an increased amount of its own signal, insulin

What is Insulin?

Effector protein that is produced by pancreatic beta cells that is able to communicate with and produce a response on target cells and lead to a DECREASE in blood glucose levels

Why are microvilli cells in the small intestine important?

They are intimately associated with small blood vessels and they absorb glucose found in intestinal tract and absorbed glucose in transported through blood vessels

True or False: Glucose metabolism is an important physiological event which leads to an increase in insulin gene transcription and mRNA translation

True! Insulin biosynthesis is regulated at both the transcriptional and translational levels.

The functional insulin protein secreted from beta cells of pancreas is made up of two chains totalling 51 amino acids. What is the difference between the chains?

Alpha chain: 21 amino acids
Beta chain: 30 amino acids

Two chains form a dimer that makes up functional insulin protein

Preproinsulin

110 amino acid precursor to functional insulin (Insulin gene encodes it)

How do N-terminal sequences assist movement of preproinsulin?

Preproinsulin contains N-terminal signal sequences which interacts with signal recognition particles to facilitate translocation of preproinsulin into lumen of the RER

How is proinsulin created?

The processing of preproinsulin through cleavage of the signal sequence which yields proinsulin

How is proinsulin further modified?

- Undergoes folding in addition to formation of 3 disulphide bonds (requires chaperone protein in RER)

Once proinsulin is folded, where is it transported?

Transported from RER to golgi apparatus where further cleavage occurs forming mature insulin dimer (releases C chain)

Phosphorylation

Reversible modification that involves the covalent attachment of a phosphate group to serine, threonine, or tyrosine residues in a protein by enzymes called kinases

Methylation

Covalent addition of methyl groups to DNA strand (Particularly promoter)

Acetylation

The addition of acetyl groups to specific amino acids

Receptors

Important proteins that receive and interpret information from such signalling molecules like insulin
- Each receptor has the ability to bind specific signals

What are receptor kinases and what is their purpose?

Insulin protein binds specific insulin receptor that fall into a family of receptors called receptor kinases (Binding of insulin to receptors enables many cells to transport glucose across plasma into cytosol)

Binding of insulin to these receptors causes a conformational change which causes the receptor monomers to pair up, leading to the activation of cytoplasmic domains of the receptor which have the ability to act like kinase proteins

Kinase proteins

Able to engage in phosphorylation of specific amino acids

What occurs as a result of intracellular signal amplification?

Extracellular insulin signal causes a series of cytoplasmic proteins to become sequentially activated and will lead to an intracellular response which is the activation of GLUCOSE TRANSPORTER PROTEINS at cell surface to increase absorption of glucose into cell

How are intracellular signals induced?

Once insulin binds to receptor, intracellular signals are induced through the activation of a series of diverse TRANSDUCER AND AMPLIFIER PROTEINS that are downstream from activated receptor

Double negative feedback

Inhibitor of the signal can also be inhibited

To produce more than one mRNA transcript from a single protein coding gene, what must occur?

Alternative splicing of pre-mRNA's must occur

Alternative splicing

Enables one pre-mRNA molecule to be spliced at different junctions to result in many different mature mRNA molecules that each contain different combinations of transcribed exons

Why might some exons be excluded during the splicing process? (This leads to the production of many isoforms of different types of mature mRNA from same pre-mRNA transcript.

This occurs because what the spliceosome will sometimes recognize as an exon in some primary transcripts, can sometimes be identified as an intron in other primary transcripts

What occurs in skeletal muscle cells that makes them have a higher affinity to insulin compared to liver cells

In skeletal muscle cells, Exon 11 is removed from mature mRNA product of gene coding for insulin receptor. In liver cells, exon 11 is attained which is why it has a lower affinity

How is the signal for insulin terminated?

After body cells have taken up glucose, there is a decrease in blood glucose that will be detected by pancreatic cells and there will be a decrease in the secretion of insulin

- The feedback of this information to the the sensor cells limits any further response in the entire system

Under favourable conditions and available nutrients, all cells have the essential machinery that favours?

Cell growth and division

Most prokaryotes grow best at?

Certain favourable temperatures

True or false, the DNA of a bacterial nucleoid contains information required to orchestrate a response to any change in the environment

True

Housekeeping Genes

a gene that is transcribed continually because its product is needed at all times and in all cells (constitutively expressed)

Include genes important for structural proteins of the cell (RNA and DNA polymerases and gene coding for ribosomal proteins)

What so housekeeping genes allow for?

They allow for the constant maintenance of general cellular activity

What are regulated Genes and when do they "turn on"

Genes that can be turned on and off on as as-needed basis

They are "turned on" when exposed to changing environment

True of false, regulated genes cannot alter the expression pattern of any genes

False, Some can alter the expression pattern of some genes

Why would things like enzymes and proteins be coded for by regulated genes?

Because the enzymes are proteins are needed to bring about changes in growth and division

If you were to expose a bacterial culture to only a limited amount of glucose, what will you find?

Once the cells use up all the glucose, bacterial growth is arrested

E. coli cells have a unique gene expression mechanism that allows them to be able to do what?

It allows them to be able to switch to metabolizing an alternative fuel source when the preferred glucose source is depleted

If E. coli cells are grown in an environment that contains glucose and lactose, when will the cell switch to utilizing lactose as a fuel source?

Bacteria will metabolize all the glucose first, before switching to lactose

True or false, it is the products of glucose metabolisms themselves that activate the switch between glucose and lactose use

True

In the absence of a direct source of glucose, the E. coli cell can metabolize the disaccharide lactose. What is lactose composed of?

Glucose and Galactose

What is the enzyme that can metabolize lactose to produce glucose and galactose called?

Beta Galactosidase enzyme

True or false, the gene that codes for B-galactosidase is constitutively expressed (always on)

False, the cell will only transcribe the gene when lactose is present and glucose is not

Francois Jacob and Jacques Monod investigated how E.coli are able to produce B-galactosidase that is needed for lactose metabolism, what did they observe?

- observed that production of the enzyme is dependent upon presence of lactose in environment
- Observed the amount of B-galactosidase protein produces by the E.coli cells began to steadily increase in response to addition of lactose (also, production of enzyme ceased when lactose was removed)

gene expression

The functional product of the gene is made, modified and activated

How many distinct levels of regulation must occur?

Three levels - Transcriptional control, translational control, post-translational control

True or false, if the transcriptional control level was disrupted, there would still be an activated protein

False, if any one of the steps is disrupted, there will be NO activated protein

Transcriptional Control

> Controls the amount of mRNA that is produced in the cell

In prokaryotes and eukaryotes, activation of transcription requires what?

Requires that proteins bind to a region near the beginning of the gene (the promoter) and increase binding of RNA polymerase - gene is now transcribed or turned on.

Translational Control

gene expression regulated by influencing the interaction of the mRNA transcripts with the ribosome

Occurs by binding of the ribosome to the 5' cap of mRNA

True or false, stability is a characteristic that determines the amount of protein produced

True, if mRNA is quickly degraded, very little protein is made

post-translational control

Regulation of gene expression by modification of proteins (e.g., addition of a phosphate group or sugar residues) after translation.
- Control mechanisms allow polypeptide chain to be folded into 3d Structure

More than a dozen post-translational modifications regulate the ability of the protein to become active or inactive by doing what?

Driving the assembly into complexes, binding of substrates, unmak enzymatic domains

Which level of regulation is the fastest?(quick cellular responses)

Post-Translational regulation is fastest as it allows the cell to have a stockpile of protein in the cell that is simply inactive
- Once the cell receives a signal, it can lead to a simple modification to turn on all of the inactive proteins

Which level of regulation is the slowest?

Transcriptional regulation is the slowest as the cell is starting from scratch
- Since cells must first transcribe, then translate and modify, it results in the delay of what is being produced

Which level of regulation is the most efficient?

Transcriptional regulation is most efficient as it does not waste any energy or resources making a mRNA or polypeptide unless it really needs to

How can efficiency be observed in E. coli cells

They only increase gene expression of B-galactosidase gene in presence of lactose as a nutrient supplement within their local environment

They are able to efficiently metabolize lactose on an as-per-needed basis

As E. coli cells utilize the nutrients that are available in the environment, the bacterial cells are able to detect two important cues that facilitate the transition from glucose to lactose metabolism. What are the cues?

Changes in glucose level and presence if lactose

Lactose Permease

Transport protein that sits in the bacterial cell membrane and allows for the transport of lactose into the bacterial cells

Beta - Galactosidase Protein

Cytoplasmically situated bacterial enzyme that cleaves the imported lactose into glucose and galactose

Operons

An advantage of the prokaryotic genome: groups of related genes with related functions can often be found clustered together

What does a bacterial operon consist of?

Promoter
Operator (on-off switch)
Coordinated gene cluster (function in common pathway or cellular response)

Operator

Sequence of nucleotides near the start of the operon that can be regulated to allow or inhibit transcription

When an operator is not bound to any transcriptional inhibitor, what happens?

RNA polymerase can attach to the promoter and transcribe the gene

What is the control of the lac operon in E.coli cells?

To regulate B-galactosidase and lactose permease expression

LacY Gene

Codes for lactose permease

LacZ Gene

codes for B-galactosidase

Lac1 gene

Controls expression of lacZ and lacY genes.