DNA, RNA, and Protein Synthesis. Molecular structure of DNA Chromosomes contain both nucleic acid...

DNA, RNA, and Protein Synthesis

Molecular structure of DNAChromosomes contain both nucleic acid and

protein.There are two types of nucleic acid, DNA and

RNA.Nucleic acid is a polymer of nucleotides, each

comprised of a ribose sugar, a phosphate group and a nitrogen containing base.

There are two types of bases, Purines and Pyrimidines.

In the 1920s, Frederick Griffith performed experiments with two strains of the bacterium Streptococcus pneumoniae.

A biological assay implicates DNA as the primary genetic molecule.

In 1952, Alfred D. Hershey and Martha Chase confirmed DNA is the genetic material

T2 bacteriophage (virus that attacks bacteria) consists of DNA core packed in protein coat

Phage DNA alone carries genetic information

A DNA nucleotide

Individual nucleotides of DNA are linked by phosphodiester bonds.

This sugar-phosphate chain forms the “backbone” of the DNA molecule.

In the 1950s, chemists Rosalind Franklin and Maurice Wilkins provided key information about DNA structure using X-ray crystallography

Erwin Chargaff noted that A = T, and G = C

James D. Watson and Francis Crick established the general structure of DNA in 1953

X-ray crystallography convinced them that the DNA molecule was helical, with certain dimensions.

Other evidence for two polynucleotide chains running antiparallel to each other

Key features of DNA:– double-stranded helix

– diameter is uniform

– the twist is right-handed

– antiparallel strands – two strands run in different directions

– Semiconservative replication uses each parent strand as template for new strand

The Meselson – Stahl experiment demonstrating replication is semi-conservative using density labeling.

DNA Replication

DNA replication is carried out by a highly complex assembly of proteins

Helicase

Single-strand binding proteinsPrimase

DNA Polymerase III

When DNA polymerase III reaches previous Okazaki fragment, it is released

DNA polymerase I replaces the primer of previous Okazaki fragment with DNA, but leaving a small “nick”

Finally, DNA ligase catalyzes formation of the phosphodiester linkage that joins the two Okazaki fragments

Denature and Renature of DNADNA double helix is stabilized by numerous

hydrogen bonds between complimentary base pairs.

DNA is denatured or melt if exposed to high temperature or extreme of pH.

The melting temperature (Tm) is the temperature at which half of the DNA molecules in a sample have been denatured.

Double helices with an access of G:C base pairs are more stable and have higher Tm than helices in which A:T base pairs predominate.

Annealing or hybridization is an essential and powerful tool

Labeled DNA fragment can be used as a probe to find its complement, even in a whole genome.

The gene of interest can be identified and isolate from DNA library.

Specific primers can be designed to amplify the gene of interest in polymerase chain reaction.

By observing the extent of annealing between DNA strands in solution, researchers can determine the degree of similarity between DNA molecules from different species.

Gene expression takes place in two steps:

Transcription – makes single-stranded RNA copy of a DNA segment

Translation – uses information encoded in RNA to make a polypeptide

http://www.anselm.edu/homepage/jpitocch/genbio/transcrtransl.JPG

Decoding Genetic Information: DNA to RNA to Protein

In normal prokaryotic and eukaryotic cells, transcription requires:

DNA template for complementary base pairing

appropriate ribonucleoside triphosphates (ATP, GTP, CTP, and UTP) to act as substrates

RNA polymerase enzyme

http://www.brooklyn.cuny.edu/bc/ahp/BioInfo/graphics/Transcription.01.GIF

Transcription

In prokaryotes, translation of mRNA often begins before transcription is complete

http://www.phschool.com/science/biology_place/biocoach/images/transcription

Figure 14.2 From Gene to Protein

Each eukaryote gene has one promoter to which RNA polymerase binds, with the help of other molecules.

At the other end of the gene there is a terminator sequence to signal end of transcription.

Transcription

In the nucleus, pre-mRNA is modified at both ends:

G cap is added at the 5′ end (modified guanosine triphosphate)—facilitates mRNA binding to ribosome.

G cap protects mRNA from being digested by ribonucleases.

Poly A tail added at 3′ end.

AAUAAA sequence after last codon is a signal for an enzyme to cut the pre-mRNA; then another enzyme adds 100 to 300 adenines—the “tail.”

May assist in export from nucleus; important for stability of mRNA.

Eukaryotic genes may have noncoding sequences—introns.

The coding sequences are exons.

Introns and exons appear in the primary mRNA transcript—pre-mRNA; introns are removed from the final mRNA.

Introns interrupt, but do not scramble, the DNA sequence that encodes a polypeptide.

Sometimes, the separated exons code for different domains (functional regions) of the protein.

Intron Splicing

Alternative Splicing

Francis Crick’s central dogma stated that information flow is: DNA codes for RNA, and RNA codes for protein

Transcription Translation

Replication

Decoding Genetic Information: DNA to RNA to Protein

Exception to the central dogma:

Viruses: Non-cellular particles that reproduce inside cells; many have RNA instead of DNA.

Viruses can replicate by transcribing from RNA to RNA, and then making multiple copies by transcription.

Other viruses such as HIV are retroviruses.

After infecting a host cell a copy of the viral genome is incorporated into the host’s genome to make more RNA.

Synthesis of DNA from RNA is reverse transcription.

http://www.brown.edu/Courses/Bio_160/Projects1999/hiv/images/Virion2.jpg

HIV, a retrovirus

TranslationConcurrent transcription and translation in prokaryotes.

TranslationTranscription and translation are spatially separated in eukaryotes.

RNA (ribonucleic acid) differs from DNA:

Usually one polynucleotide strand

The sugar is ribose

Contains uracil (U) instead of thymine (T)

Bases in RNA can pair with a single strand of DNA, except that adenine pairs with uracil instead of thymine.

Single-strand RNA can fold into complex shapes by internal base pairing.

Three kinds of RNA in protein synthesis:Messenger RNA (mRNA)—carries copy of a

DNA sequence to site of protein synthesis at the ribosome

Transfer RNA (tRNA)—carries amino acids for polypeptide assembly

Ribosomal RNA (rRNA)—catalyzes peptide bonds and provides structure

Messenger RNA (mRNA) Produced by transcription as a

complementary copy of DNAmoves from nucleus of

eukaryotic cells into cytoplasmserves as template for protein

synthesis

tRNA – the “adapter” moleculerequired to assure specificity in translation of mRNA into

proteinstRNAs must read mRNA correctlySpecific tRNAs must carry correct amino acids

tRNAs are “adapters”link a specific codon with

specific amino acid

tRNA has three functions:

carries an amino acid

associates with mRNA molecules

interacts with ribosomes

Amino acid binding site at 3’ end of every tRNA (for covalently binding a specific amino acid)

Anticodon On other side of the molecule three bases which are complementary

to appropriate codon The codon and anticodon unite by

complementary base pairing.

http://www.wiley.com/legacy/college/boyer/0470003790/structure/tRNA/trna_diagram.gif

WobbleSpecificity for base at codon’s third

position is not always observed Ex: codons for alanine — GCA, GCC,

and GCU — are recognized by the same tRNA

Allows cells to produce fewer tRNA species; but not in all cases — the genetic code remains unambiguous

http://www.wiley.com/legacy/college/boyer/0470003790/structure/tRNA/trna_diagram.gif

Aminoacyl-tRNA synthetases Attaches amino acids to tRNAsTheir three-part active sites bind:

a specific amino acid ATP a specific tRNA, charged with a high-energy bond

High-energy bond provides energy for making a peptide bond

Translation also occurs in three steps:

InitiationElongationTermination

http://srv2.lycoming.edu/~newman/courses/bio43704/ribosome/ribosome1.jpg

InitiationStart codon (AUG)

designates first amino acid (methionine) in all proteins

Large subunit then joins complex with tRNA at P-site, all guided by initiation factors

InitiationThe mRNA binds to the small

ribosomal subunit.The Shine-Dalgarno

sequence near the 5’ end of the mRNA base pairs with a sequence near the 3’ end of the rRNA of the small ribosomal subunit.

Elongation: The second charged tRNA enters the A site.

Large subunit catalyzes two reactions:It breaks bond between tRNA in P site and its

amino acidPeptide bond forms between that amino acid

and the amino acid on tRNA in the A siteWhen the first tRNA has released its methionine,

it moves to the E site and dissociates from the ribosome—can then become charged again.

Elongation occurs as the steps are repeated, assisted by proteins called elongation factors.

TerminationWhen stop codon is

reached…Release factor and a

water molecule enter A site, instead of an amino acid

Newly completed protein separates from ribosome, and subunits separate

Protein modifications:Proteolysis: Cutting of a long polypeptide

chain into final products, by proteasesGlycosylation: Addition of sugars to form

glycoproteinsPhosphorylation: Addition of phosphate

groups catalyzed by protein kinases— charged phosphate groups change the conformation

High accuracy of protein synthesis

Mutation Results in Mutant Proteins

The genetic disease, sickle-cell anemia, caused by mutant proteins with single amino acid replacement. A single amino acid change in such a large complex hemoglobin could produce profound changes in activity.

Control of Gene Expression

Cells conserve energy and resources by making proteins only when needed.

Intricate molecular mechanisms must exist in cells to control the numbers of their many proteins.

There are three ways that might achieve this differential synthesis: transcriptional control, translational control, and post-transcriptional control.

Cells regulate protein synthesis at any of four pointsControlling the rate of mRNA synthesisThe stability of mRNAsChanging the rate of protein synthesisThe stability of proteins


Cells regulate protein synthesis by several methods:Block transcription of the gene Hydrolyze the mRNA after it is madePrevent translation of mRNA at the ribosomeHydrolyze the protein after it is madeInhibit the function of the protein


Positive regulation Gene is not normally transcribed (usually “off”)Activator protein binds to stimulate transcription

Regulation of mRNA Transcription

One of two types of regulatory protein binds to promoter :Negative regulation

Gene is normally transcribed (usually “on”) Binding of a repressor protein prevents

transcription


E. coli prefers glucose as an energy source, but can use lactose if glucose is low.

3 enzymes are required for lactose metabolism

presence of lactose stimulates production of these enzymes

lactose is an inducer

http://spacebio.net/modules

E. coli


http://spacebio.net/modules

• enzymes that are produced in the presence of an inducer are said to be inducible

• enzymes that are made all the time are said to be constitutive

E. coli


Operon is the whole unitpromoter, operator, and one or more structural

genesOperon containing genes for lactose metabolism:

lac operon


Structural genes specify primary protein structure — the amino acid sequenceThe three structural genes for lactose enzymes are

adjacent on chromosome They share a promoter, and are transcribed together


Prokaryotes shut down transcription by placing obstacle between promoter and structural genea protein called a repressor bind to operator—

blocks transcription of mRNA


Regulation of lac Operon

A repressor protein is coded by a regulatory gene.The regulatory gene that codes for the lac repressor is

the i (inducibility) gene

i gene is near lac structural genes, but not all regulatory genes are near their operons


Regulatory genes like i have their own promoter, called pi

The i gene is expressed constitutively (expression is constant).


The repressor protein has two binding sitesone for the operatorone for inducer (lactose)

Operator binding site


Binding inducer to repressor, changes repressor shape allows promoter to bind RNA polymeraseWhen lactose concentration drops, inducers separate

from repressors — repressor again binds operator, transcriptions stops


• Promoter is the specific region of the DNA that bound to RNA polymerase for efficient transcription.• Two highly conserved, separate nucleotide blocks make up the promoter of the E. coli lac operon.• These blocked are designated -10 and -35 upstream of the mRNA start site +1.•Mutations in these regions have mild and severe effects on transcription.

The sequences required for RNA polymerase binding and initiation are contained within less than 40 nucleotides.The initial step is the recognition and binding of an

RNA polymerase to the -35 region.Subsequently, the -10 region melts into its

component single strands allowing transcription to begin at +1 position.

Bacterial RNA polymerase core complexes are bound by a single initiation factor σ (sigma).

E coli can control gene expression by activation of σ factor in response to stimuli.σ 70 is the most common initiation factor in E. coli genome

that binds to many other promoters besides lac promoter.σ 32 is activated in response to “heat shock” and directs

polymerase to transcribe specific genes to respond to this stress.

σ 54 is activated when cells are starved for nitrogen, turning on genes that are responsible for uptake of nitrogen.

Sigma factors produced by E. coli

Genes are activated when transcription factors bind specific DNA sequences near promoterIf both glucose and lactose

are present, E. coli will first use glucose.

Presence of glucose lowers concentration of cAMP

thus less CRP binding to promoter, resulting in less efficient transcription of lactose-metabolizing enzymes

When glucose is used up, starvation signals accumulate in the cell, and trigger an activation of lac operon.lac operon can increase efficiency

of the promoterA regulatory protein CRP binds

cAMP This complex binds to DNA just

upstream of promoter. Allows more efficient binding of

RNA polymerase to promoter

A protein is repressible if synthesis can be turned off by a biochemical cue (e.g., ample supply of that protein)The trp operon controls

synthesis of tryptophan — it is a repressible system

Gene is normally “on”, transcribing mRNA and synthesizing structural proteins

Regulation of trp Operon

The repressor must first bind with a corepressor, in this case tryptophan (when its abundant), activating repressor

Active repressor turns gene “off”, blocking transcription

Regulation of trp Operon

Inducible systems (lac operon) Substrate of a metabolic

pathway (inducer) interacts with a regulatory protein (repressor) — repressor cannot bind to operator —allowing transcription

Control catabolic pathways (turned on when substrate, lactose, is present)

lac Operon VS trp Operon

Repressible system (trp operon)Product of a metabolic pathway

(corepressor) interacts with a regulatory protein (repressor) allowing it to bind to operator, blocking transcription

Control anabolic pathways (turned on when product, tryptophan, is not present).

lac Operon VS trp Operon

The Attenuation in trp operon

• Attenuation is a method of regulating transcription by preventing complete mRNA synthesis.– The 5’ end of trp operon mRNA, the leader region (region 1) is rich in

tryptophan codon.– When tryptophan is available, the translation of this region occurs.– As this happens, the trp mRNA forms a stem-loop structure between

region 3 and 4, transcription is attenuated.– When the tryptophan level drops, the attenuation is relieved.– The ribosome is stalled at the leader sequence, different stem-loop

forms (2 and 3), allows the transcription to continue.

DNA, RNA, and Protein Synthesis. Molecular structure of DNA Chromosomes contain both nucleic acid...

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