transcription and translation lecture notes

© 2011 Pearson Education, Inc.

Lectures by Stephanie Scher Pandolfi

BIOLOGICAL SCIENCE FOURTH EDITION

SCOTT FREEMAN

16Transcription

and Translation

Review of previous chapter- what are two linked genes? ; They are on the same chromosome

Start chapter lecture -

Transcription is the creation of a piece of RNA based on the information in DNA .What carries out translation? ; Ribosomes. Ribosomes read the mRNA

This will be the last chapter***The main focus of this chapter-•The details of transcription and translation. •The differences between prokaryotes and eukaryotes

Jorge Pinzon

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Key Concepts

After RNA polymerase binds DNA with the help of other proteins, it catalyzes the production of an RNA molecule whose base sequence is complementary to the base sequence of the DNA template strand.

Eukaryotic genes contain regions called exons and regions called introns; during RNA processing, the regions coded by introns are removed, and the ends of the RNA receive a cap and tail.

Key points –•mRNA in eukaryotes are modified in 3 ways 1.Cap added to the 5’ prime end 2.Poly A tail is added to the 3’ prime end 3.Splicing- Exons spliced/ together

*Reason- stabilize RNA to last longer. Cap gives protection in order for to stay longer because they have to leave nucleus before being translated


Board drawing splicing


Key Concepts

Ribosomes translate mRNAs into proteins with the help of intermediary molecules called transfer RNAs (tRNAs).

Each transfer RNA carries an amino acid corresponding to the tRNA’s three-base-long anticodon.

In the ribosome, the tRNA anticodon binds to a three-base-long mRNA codon, causing the amino acid carried by the transfer RNA to be added to the growing protein.

The codons recruit each amino acid by means of tRNA•There are 20 tRNAs•They are anticodons that compliment the codons


Introduction• A cell builds the proteins it needs from instructions encoded in its

genome according to the central dogma of molecular biology.


Overview of Transcription

• The first step in converting genetic information into proteins is transcription, the synthesis of an mRNA version of the instructions stored in DNA.

• RNA polymerase performs this synthesis by transcribing only one strand of DNA, called the template strand.

• The other DNA strand is called the non-template, or coding

strand, which matches the sequence of the mRNA, except that RNA has uracil (U) in place of thymine (T).


Characteristics of RNA Polymerase

Like the DNA polymerases, an RNA polymerase performs a template-directed synthesis in the 5′ to 3′ direction. But unlike DNA polymerases, RNA polymerases do not require a primer to begin transcription. Because they string RNA together

• Bacteria have one RNA polymerase while eukaryotes have three distinct types, RNA polymerase I, II, and III.


Initiation: How Does Transcription Begin?

• Initiation is the first phase of transcription.– However, RNA polymerase cannot initiate transcription on its

own.– Sigma, a protein subunit, must first bind to the polymerase.

In prokaryotic transcription is initiated when Sigma recognizes special sequences in DNA helps RNA polymerase where to start


What Role Does Sigma Play in Initiation?

• Sigma and RNA polymerase together form a holoenzyme, an enzyme made up of a core enzyme and other required proteins.

• Prokaryotic RNA polymerase is a holoenzyme made up of the core enzyme, which has the ability to synthesize RNA, and a sigma subunit.

– Sigma acts as a regulatory factor, guiding RNA polymerase to specific promoter sequences on the DNA template strand.

Promoter sequences in DNA direct RNA polymerase where to transcribe gene


Bacterial Promoters• Bacterial promoters are comprised of 4050 base pairs and have two

key regions.

• The –10 box is found 10 bases upstream (in the opposite direction of RNA polymerase movement during transcription) from the transcription start site (the +1 site) and consists of the sequence TATAAT.

• The –35 box, consisting of the sequence TTGACA, is 35 bases upstream from the +1 site.

• All bacterial promoters have a –10 box and a –35 box, the remainder of the promoter sequence varies.

***slide only used to explain


Eukaryotic Promoters• Eukaryotes have a much more diverse and complex series of

promoters than do prokaryotes.

• Many of the eukaryotic promoters include a unique sequence called the TATA box, centered about 30 base pairs upstream of the transcription start site.

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In Bacteria, Sigma Subunits Initiate Transcription

• Transcription begins when sigma, as part of the holoenzyme complex, binds to the –35 and –10 boxes.

• Sigma, and not RNA polymerase, makes the initial contact with DNA that starts transcription, supporting the hypothesis that sigma is a regulatory protein.

• Most bacteria have several types of sigma proteins.– Each type allows RNA polymerase to bind to a different type

of promoter and therefore a different kind of gene.

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Transcription Initiation in Eukaryotes• As with bacteria, the RNA polymerase does not bind directly to the promoter.

• In eukaryotes, a group of proteins called basal transcription factors bind to the DNA promoter, thus initiating transcription. Need to know basal transcription factors***

• Basal transcription factors perform a similar function to bacterial sigma proteins.


What Occurs Inside the Holoenzyme?

• Sigma opens the DNA double helix and the template strand is threaded through the RNA polymerase active site.

• An incoming ribonucleoside triphosphate (NTP) pairs with a complementary base on the DNA template strand, and RNA polymerization begins.

• Sigma dissociates from the core enzyme once the initiation phase of transcription is completed.

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1. Where transcription starts, in promoter sequence from –10box to -35box by polymerase binding to sigma at the +1 site

2. Opens bubble in DNA to string together NTPS

3. In Eukaryotes there is no sigma instead transcription factors

4. Here ribosomes can start synthesizing mRNA as soon as it is made be because there is no nuclear envelope 4

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Elongation and Termination

• During the elongation phase of transcription, RNA polymerase moves along the DNA template and synthesizes RNA in the 5' 3' direction. Polymerase continues down DNA to string together nucleotides

• Transcription ends with a termination phase. In this phase, RNA polymerase encounters a transcription termination signal in the DNA template.

• In bacteria the transcription termination signal codes for RNA forming a hairpin structure, which causes the RNA polymerase to separate from the RNA transcript, ending transcription.


Transcription

Video Does not show the 3 modifications in mRNA before leaving nucleus


RNA Synthesis

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RNA Processing in Eukaryotes

• In bacteria, the information in DNA is converted to mRNA directly. In eukaryotes, however, the product of transcription is an immature primary transcript, or pre-mRNA. Before primary transcripts can be translated, they have to be processed in a complex series of steps.

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The Discovery of Eukaryotic Genes in Pieces

• The protein-coding regions of eukaryotic genes are interrupted by noncoding regions.

– To make a functional mRNA, these noncoding regions must be removed.

• Exons are the coding regions of eukaryotic genes that will be part of the final mRNA product. Exons stay

• The intervening noncoding sequences are called introns, and are not in the final mRNA. Introns Taken out

• Eukaryotic genes are much larger than their corresponding mature mRNA.


RNA Splicing

• The transcription of eukaryotic genes by RNA polymerase generates a primary RNA transcript that contains exons and introns.

– Introns are removed by splicing.

• Small nuclear ribonucleoproteins (snRNPs) form a complex called a spliceosome. This spliceosome catalyzes the splicing reaction.


Adding Caps and Tails to RNA Transcripts

• Primary RNA transcripts are also processed by the addition of a 5′ cap and a poly(A) tail.

With the addition of cap and tail and completion of splicing, processing of the primary RNA transcript is complete. The product is a mature mRNA.

• The 5' cap serves as a recognition signal for the translation machinery.

• The poly(A) tail extends the life of an mRNA by protecting it from degradation.

Above bullets review modification of mRNA


Another example of cap and tail.


An Introduction to Translation

• In translation, the sequence of bases in the mRNA is converted to an amino acid sequence in a protein.

• Ribosomes catalyze translation of the mRNA sequence into protein.

Review of past information


Transcription and Translation in Bacteria

• In bacteria, transcription and translation can occur simultaneously. Bacterial ribosomes begin translating an mRNA before RNA polymerase has finished transcribing it.

– Multiple ribosomes attached to an mRNA form a polyribosome. Poly: many

– Many ribosomes are simultaneously working on mRNA– Description in next slide***

• In eukaryotes, transcription and translation are separated. mRNAs are synthesized and processed in the nucleus and then transported to the cytoplasm for translation by ribosomes.


What type of cell is this Eukaryote or prokaryote ?

Prokaryote, because the ribosomes is able to attach to mrna while being transcribed.Nuclear envelope prevents this in Eukaryotes


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Transcription and Translation in Eukaryotes

• In eukaryotes, transcription and translation are separated. mRNAs are synthesized and processed in the nucleus and then transported to the cytoplasm for translation by ribosomes.


How Does an mRNA Triplet Specify an Amino Acid?

• There were two hypotheses regarding the specification of amino acid sequence by a sequence of nucleotide bases:

1. mRNA codons and amino acids interact directly.2. Francis Crick proposed that an adapter molecule holds amino

acids in place while interacting directly and specifically with a codon in mRNA.

• The adapter molecule was later found to be a small RNA called transfer RNA (tRNA).


The Characteristics of Transfer RNA

• ATP is required to attach tRNA to an amino acid.

• Enzymes called aminoacyl tRNA synthetases “charge” the tRNA by catalyzing the addition of amino acids to tRNAs.

• For each of the 20 amino acids, there is a different aminoacyl tRNA synthetase and one or more tRNAs.

• A tRNA covalently linked to its corresponding amino acid is called an aminoacyl tRNA.

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What Happens to the Amino Acids Attached to tRNA?

• Experiments with radioactive amino acids revealed that they are lost from tRNAs and incorporated into polypeptides synthesized in ribosomes.

These results inspired the use of “transfer” in tRNA’s name, because amino acids are transferred from the RNA to the growing end of a new polypeptide. The experiment also confirmed that aminoacyl tRNAs act as the interpreter in the translation process: tRNAs are Crick’s adapter molecules.

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What Do tRNAs Look Like?

• The CCA sequence at the 3' end of each tRNA is the binding site for amino acids.

• The triplet on the loop at the opposite end is the anticodon that base pairs with the mRNA codon. Important***

• The secondary structure of tRNA folds over to produce an L-shaped tertiary structure.

All of the tRNAs in a cell have the same structure, shaped like an upside-down L. They vary at the anticodon and attached amino acid.


Reason why anticodons are important, it lets the Ribosome know that this codon should bring this amino acid. The amino acid is attached to this particular tRNA that has anticodon to compliment this codon.


How Many tRNAs Are There?

• There are 61 different codons but only about 40 tRNAs in most cells.

• To resolve this deficit, Francis Crick proposed the wobble hypothesis. This hypothesis proposes that the anticodon of tRNAs can still bind successfully to a codon whose third position requires a nonstandard base pairing.

• Thus, one tRNA is able to base pair with more than one type of codon.


The Structure and Function of Ribosomes

• Ribosomes contain protein and ribosomal RNA (rRNA).

• Ribosomes can be separated into two subunits: – The small subunit, which holds the mRNA in place during

translation.– The large subunit, where peptide bonds form.

• During translation, three distinct tRNAs line up within the ribosome.


Ribosomes and the Mechanism of Translation

• All three tRNAs are bound at their anticodons to the corresponding mRNA codon.

• There are 3 chambers inside the ribosome – The A site of the ribosome is the acceptor site for an

aminoacyl tRNA. Acceptor of amino acid

– The P site is where a peptide bond forms that adds an amino acid to the growing polypeptide chain. Polypeptide- where they form

– The E site is where tRNAs no longer bound to an amino acid exit the ribosome. Exit


Proccess will continue until a stop codon is reached.Stop codon recognized by release factor protein. Slide 69***


Ribosomes and the Mechanism of Translation

The ribosome is a molecular machine that synthesizes proteins in a three-step sequence.1. An aminoacyl tRNA carrying the correct anticodon for the

mRNA codon enters the A site.2. A peptide bond forms between the amino acid on the

aminoacyl tRNA in the A site and the growing polypeptide on the tRNA in the P site.

3. The ribosome moves ahead three bases and all three tRNAs move down one position; the tRNA in the E site exits.


The Phases of Translation

• Translation has three phases: initiation, elongation, and termination.


Initiation

• The initiation phase of translation begins at the AUG start codon.• ***there are many AUG codons• In bacteria, the start codon is preceded by a ribosome binding site

(also called the Shine-Dalgarno sequence) that is complementary to a section of one rRNA in the small ribosomal subunit. The Shine-Dalgarno sequence follows the proper start codon in for the ribosome to know where to start in Prokaryotes cells

• Eukaryotes use a sequence called a kozak sequence***

• The interaction between the small subunit and the mRNA is mediated by initiation factors.


Initiation in Bacteria

• Translation initiation is a three-step process in bacteria: 1. The mRNA binds to a small ribosomal subunit.2. The initiator aminoacyl tRNA bearing N-formylmethionine

(f-met) binds to the start codon.3. The large ribosomal subunit binds, completing the complex.

• Translation is now ready to begin.


Elongation

• At the start of the elongation phase, the initiator tRNA is in the P site, and the E and A sites are empty.

• An aminoacyl tRNA binds to the codon in the A site via complementary base pairing between anticodon and codon.

• Peptide bonds form between amino acids on the tRNAs in the P and A sites.

– After peptide bond formation, the polypeptide on the tRNA in the P site is transferred to the tRNA in the A site.


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Is the Ribosome an Enzyme or a Ribozyme?

• The active site of the ribosome is entirely ribosomal RNA.

• Thus, ribosomal RNA catalyzes peptide bond formation and the ribosome is a ribozyme.


Moving Down the mRNA

• Translocation occurs when elongation factors move the mRNA down the ribosome three nucleotides at a time, and the tRNA attached to the growing protein moves into the P site.

• The A site is now available to accept a new aminoacyl tRNA for binding to the next codon.

• The tRNA that was in the P site moves to the E site, and if the E site is occupied, that tRNA is ejected.


Elongation

• Elongation has three steps:1. Arrival of the aminoacyl tRNA.2. Peptide bond formation.3. Translocation.


Termination

• The termination phase starts when the A site encounters a stop codon.

• This causes a protein called a release factor to enter the site. Release factors resemble tRNAs in size and shape but do not carry an amino acid.

• These factors catalyze hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain.

• Proccess will continue until a stop codon is reached.• Stop codon recognized by release factor protein. Slide 69***


Release factor has no anticodon.It is a protein causes everything to be released (like a bomb) and stopping translation


TranslationSkip slide


Synthesizing Proteins

Video viewed in class lecture


Post-Translational Modifications

• Most proteins go through an extensive series of processing steps, collectively called post-translational modification, before they are ready to go to work in a cell.

• Molecular chaperones speed folding of the protein. Folding determines a protein's shape and therefore its function. From previous chapters we learned how after translation chaperones and folding are important.

• Many proteins are altered by enzymes that add or remove a phosphate group. These changes often switch the protein from an inactive state to an active state or vice versa.