Chapter 17 – from gene to protein - PowerPoint Presentation

Slide1

Chapter 17 – from gene to protein

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The information content of genes is in the form of specific sequences of nucleotides along the DNA

strands. The

DNA of an organism leads to specific traits by dictating the synthesis of proteins and of RNA molecules involved in protein synthesis (

gene expression

.)

Proteins

are the link between genotype and phenotype

.

Slide2

Archibald garrod

The study of metabolic defects provided evidence that genes specify proteins

.

Garrod

discovered

that proteins (enzymes) are the link between genotype and phenotype.

He figured out that some inherited diseases are the

inability to make enzymes

He noticed that the diaper of a baby was very brown. He determined that the baby had

alkaptonuria, which is a recessively inherited disorder where the urine is a brown color. This is due to homogentisic acid which cannot be broken down in the body, so it is excreted in the urine. The reason it cannot be broken down is because there is an absence of the enzyme needed in the biochemical pathway.

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Slide3

Beadle and

tatum

Beadle and Tatum showed the relationship between genes and enzymes

. They used the bread mold

Neurospora

and exposed it to X-rays to get mutants. They found 3 different classes of mutants. Each mutant was defective in a different gene. They exposed these mutants to different environments to see which ones allowed arginine to grow. They deduced that each mutant was unable to carry out one step in the arginine pathway – probably because it lacked the necessary enzyme

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One gene – one enzyme

hypothesis…and the evolution of that hypothesis

From Beadle and Tatums experiments, they came up with the one gene, one enzyme hypothesis.

However, not all proteins are enzymes, so it became the one gene- one protein hypothesis

.

BUT…some genes have more than one polypeptide (THINK: quaternary structure of proteins), so it

led to the

one gene- one polypeptide hypothesis

. The newest discoveries have been taken into consideration and the scientific community have updated the definition of a gene as: A gene is a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule.4

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Overview: transcription translation

Transcription = DNA → RNA

Translation = RNA → Protein

DNA

transcription

Primary transcript (pre-mRNA)

RNA processing

mRNA

translation protein

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Genes provide the instructions for making specific proteins and getting from gene to protein needs two stages:

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Transcription and Translation in prokaryotes vs. eukaryotes

The basic mechanics of transcription and translation are similar in eukaryotes and bacteria.

Bacteria lack nuclei, and their DNA is not separated from ribosomes and other protein-synthesizing equipment.

This allows the coupling of transcription and translation.

In a eukaryotic cell, transcription occurs in the

nucleus

, and translation occurs at

ribosomes

in the cytoplasm.6The molecular chain of command in a cell has a directional flow of genetic information:

DNA  RNA  proteinFrancis Crick dubbed this concept the central dogma in 1956.

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Coding for amino acids

The message is carried in RNA in the form of

codons

(3 bases). It is read in the 5’ → 3’ direction.

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The triplets (codons) code for the specific amino acids

With

a triplet code

, three consecutive bases specify an amino acid, creating 43

(64) possible code words

.

During transcription, one DNA strand, the

template strand

, provides a template for ordering the sequence of nucleotide bases in an mRNA transcript.The mRNA base triplets are called codons. Each codon specifies which one of the 20 amino acids will be incorporated at the corresponding position along a polypeptide chain.The starting point establishes the reading frame

; subsequent codons are read in groups of three nucleotides.8

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Nirenberg determined

the first match: UUU codes for the amino acid

phenylalanine.

Sixty-one

of 64 triplets code for amino

acids.

The

codon

AUG not only codes for the amino acid methionine but also indicates the “start” or initiation of translation.

Three codons do not indicate amino acids but are “stop” signals marking the termination of translation.9

Marshall Nirenberg

deciphered the code for the amino acids in 1961.

The genetic code must have

evolved very early in the history of

life



It is

nearly universal, shared by organisms from the simplest bacteria to the most complex plants and animals

.

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transcription

Promoter

– DNA sequence where RNA attaches and initiates transcription

Terminator

– sequence that signals the end of transcription

Transcription Unit

– sequence of DNA that is transcribed into RNA

DNA → RNA

3 Steps of Transcription: 1. Initiation 2. Elongation 3. Termination

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Transcription - Initiation

The

promoter

determines which strand is the

template

and then

transcription factors

help RNA polymerase bind. The TATA box is an important part of the promoter that helps initiate transcription. The transcription complex consists of the promoter, transcription factors, and RNA polymerase.11

RNA polymerase separates the DNA strands at the appropriate point and joins RNA nucleotides complementary to the DNA template strand. Like DNA polymerases, RNA polymerases can assemble a polynucleotide only in its 53 direction (therefore the template strand is 3’ 

5

’

.)

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Transcription - elongation

The RNA polymerase adds RNA nucleotides about

10-20

at a

time

to the growing 3’ end.

Several mRNA strands can be made at the same time….several different RNA polymerases can all be on the same DNA molecule and can all create

mRNA. This helps the cell make the encoded protein in large amounts. 12

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Transcription - termination

At this point, transcription has given us the primary transcripts or

pre-mRNA

In prokaryotes, termination stops at the termination signal (end of the gene)

In eukaryotes, transcription continues for

10-35

nucleotides past the stop signal. Later in the process, it gets cut down.

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RNA Processing:

Modifying the pre-mrna

- At the

5’ end

, a

5’ cap

is added (which is a modified guanine molecule)

At

the 3’ end, there is the poly-A tail (50-250 adenine nucleotides) 

functions in helping to inhibit degradation and helps exportation from nucleus)- Both of these modifications have several important functions: Exporting mRNA from the nucleusProtecting mRNA from hydrolytic enzymesHelping the ribosome attach to the 5’ end of the mRNA14

After both ends are modified, the

introns

(non-coding portions) are spliced out.

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RNA Splicing

The introns are cut out using

splicesomes

. Therefore, the mRNA that leaves the nucleus (exons only) is the abridged version that only carries genes – not “filler” DNA.

Introns

= non-coding segments

Exons

= coding segments

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Slide16

RNA splicing technique - splicesomes

There are short sequences at the end of introns that signal to the

snRNP’s

(small nuclear

ribonucleoproteins

). The

snRNP’s

recognize these sites and the splicesomes then cut out the introns and reattach the exons. Ribozyme → RNA molecules that act like enzymes; in some organsisms RNA splicing can occur without additional proteins because the

introns can catalyze their own excision16

Slide17

Alternative rna

splicing

Humans can get along with a small number of genes because we can “shuffle” our DNA; different polypeptides can be made depending on which segments we consider introns and which are considered exons.

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Translation – from RNA to protein

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BUILDING A POLYPEPTIDE!

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tRNA -

TRANSLATOR

The cell is always stocked with all 20 AA’s (from diet)

The tRNA is folded like a cloverleaf; on one side it has an

anticodon

that matches up with the codon from the mRNA; on the other side it carries a specific AA

The

tRNA’s

are used over and over; they drop off their AA’s and then go get another to be used again

Wobble → relaxation of 3rd base pairing; sometimes the 3rd base of the ANTICODON has an “I” (inosine), which is an altered adenine; this can match up with U, C, or A; If each anticodon had to be a perfect match to each codon, we would expect to find 61 types of tRNA, but the actual number is about 45, because the anticodons of some tRNAs

recognize more than one

codon (the wobble!!)

CCI anticodon can match up with GGU, GGC and GGA (codons)

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Slide20

Aminoacyl-

tRNA synthetase

This enzyme attaches each AA to its appropriate tRNA.

This process uses 1 ATP

There are 20 different aminoacyl-tRNA synthetases (one for each AA)

Process

:

The active site of the aminoacyl tRNA synthetase binds to the AA and ATP

The ATP loses 2 P groups to become AMP and binds with the AAThen the right tRNA binds to the AA and displaces the AMPThe enzyme then releases the “activated AA”

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Ribosomes:

Sites of Translation

Ribosomes consist of 2 subunits, large and small; they are composed of rRNA and proteins

They have 3 binding sites for tRNA:

E

= about to exit

P

= holds the AA chain A = “on-deck” AAThe ribosomes itself catalyzes the peptide bond between amino acids. 21

Like transcription, translation can be divided into 3 stages:

-

initiation

- elongation

- termination

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Energy source for translation

GTP (

guanosine

triphosphate

) → energy source for translation; this is very similar to ATP and releases energy by breaking off phosphates

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Slide23

Translation - initiation

Steps

:

1. Small ribosomal subunit binds to mRNA leader (5’ end)

2. Initiator tRNA (

methionine

) binds to “start” codon –

AUG

3. Next the large ribosomal subunit binds 4. All of these components (small unit, mRNA, tRNA, large subunit) are brought together by initiation factors and form the translation initiation complex

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Slide24

Translation - elongation

3 step process for each AA:

1. Codon

recognition

2. Peptide

bond formation

3. Translocation

This process uses

elongation factors (proteins)24

Slide25

Translation - termination

When a stop

codon (mRNA) gets to get the A-site and instead of a tRNA binding, a

release factor

binds. This adds a

water molecule

to the AA chain, and then releases the chain from the ribosome.

After the chain is released, all the factors dissociate from one another.

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Overview – protein synthesis

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Slide27

Polyribosomes

or polysomes

This is when many ribosomes trail along the same mRNA molecule. They can translate many proteins simultaneously and therefore are much more efficient.

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Post-translational modifications

During and after synthesis, a polypeptide spontaneously

coils and folds to its three-dimensional shape.In addition, proteins may require post-translational modifications

before doing their particular job.These modifications may require additions such as sugars, lipids, or phosphate groups to amino acids.In other cases, a polypeptide may be cleaved in two or more pieces OR two or more polypeptides may join to form a protein with quaternary structure

.

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Signal peptides – determine whether ribosome will be attached or free

All ribosomes start as free (in the cytosol

); however, the polypeptide can cue the ribosome to go attach to the ER and become bound. The

signal peptide

is a sequence of

about 20 AA’s

near the front of the strand that tells the ribosome to go attach. This is the case for proteins/ enzymes that

are going to be secreted from the cell

. The signal recognition particle (SRP) sees this signal peptide and brings the ribosome to the ER to attach.

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Slide30

mutations

A

point

mutation (also called a

substitution

)

is a change in

one base pair

. It can have huge effects (sickle cell) or no affect at all (silent mutation), depending on which base is affected and where the AA is located in the protein. 30Mutations are changes in the genetic material of a cell (or virus). They are the ultimate source of new genes (and genetic diversity

!)

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Missense

and nonsense mutations

Missense

= codes for a different AA

Nonsense

= changes into a stop codon, so it leads to a nonfunctional

protein

Silent

= changes the nucleotide but it codes for the same AA

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Slide32

Frameshift mutations

A

frameshift

mutation is when there is an

insertion

or

deletion

that causes the reading frame to change. This means that all of the AA’s after the mutation will be wrong. It has disastrous effects.

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Slide33

mutations can occur during DNA replication, DNA repair, or DNA

recombination

Errors during DNA replication or recombination can lead to nucleotide-pair substitutions, insertions, or deletions.

Mutagens are chemical or physical agents that interact with DNA to cause mutations.Physical agents include high-energy radiation like X-rays and ultraviolet light.Chemical mutagens cause mutations in different ways.

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