Gene Expression: DNA to Protein

Initiating Translation

- A section of rRNA in a small ribosomal subunit binds to a complementary sequence on an mRNA. The mRNA region is called the ribosome binding site (Shine-Dalgarno sequence). This site is about 6 nucleotides upstream from the start codon. - The interactions between the small subunit, the mRNA, and the tRNA are mediated by initiation factors. Initiation factors help in preparing the ribosome for translation, including binding the first aminoacyl tRNA to the ribosome. IN bacteria, the initiator tRNA bears a modified form of methionine (f-met). In eukaryotes, the initiating tRNA carries a normal methionine. - Initiation is complete when the large subunit joins the complex. When the ribosome is completely assembled, the tRNA bearing f-met or Met occupies the P-site. To summarize (1) mRNA binds to a small ribosomal unit. (2) the initiator aminoacyl tRNA bearing f-met or Met binds to the start codon. (3) the large ribosomal subunit binds, completing the complex.

RNA Processing consists of

- Adding cap (modified guanine with three phosphates) to the 5' end of the pre-mRNA -Adding a poly(A) tail (100-250 adenine nucleotide sequence) to the 3' end of the pre-mRNA -splicing out the introns and linking the exons together with phosphodiester linkage

How are amino acids attached to tRNA?

- An input of energy, in the form of ATP, is required to attach an amino acid to a tRNA - Enzymes called amino-tRNA synthetases catalyze the addition of animo acids to the tRNA. - For each of the 20 major amino acids, there is a different aminoacyl-tRNA synthetase and one or more tRNAs Each aminoacyl-tRNA synthetase has a binding site for a particular amino acid and a particular tRNA. Subtle differences in tRNA shape and base sequence allow the enzyme to recognize the correct tRNA for the correct amino acid

Chromosome mutations

- Changes in chromosome number - poylploidy; aneuploidy - Changes in the composition of individual chromosomes - inversion - A mutation in which a segment of a chromosome, flips, ans rejoins in reverse orientation - translocation - A type of mutation in which a piece of a chromosome moves to a nonhomologous chromosome - deletion - loss of a part of a chromosome - duplication - An additional copy of a part of a chromosome

Protein synthesis in a ribosome

- The tRNA caries the amino acid. The tRNA's position in the ribosome is called the A site - for acceptor or aminoacyl - The tRNA that is in the middle holds the growing polypeptide chain and occupies the P site (for peptidyl, inside the ribosome. - The tRNA no longer has an amino acid attached and is about to leave the ribosome. This tRNA occupies the E site (exit) 1. An aminoacyl tRNA diffuses into the A site; if its anticodon matches a codon in mRNA, it stays in the ribosome. 2. A peptide bond forms between the amino acid held by the aminoacyl tRNA in the A site and the growing polypeptide, which was held by a tRNA in the P site 3. The ribosome moves down the mRNA by one codon, and all three tRNAs move one position within the ribosome. The tRNA in the E site exits; the tRNA in the P site moves to the E site; and the tRNA in the A site switches to the P site. The protein being synthesized grows by one amino acid at a time each time the three-step process repeats. Protein synthesis starts at the N-terminus of the polypeptide and proceeds to the carbonyl end (C-terminus)

Terminating Translation

- When the translocating ribosome reaches one of the stop codons, a protein called a release factor recognizes the stop codon and fills the A site. Release factors fit tightly into the A site because they have the size and shape of a tRNA coming into the ribosome. When a release factor occupies the A site, the proteins active site catalyzes the hydrolysis of the bond that links the tRNA in the P site to the polypeptide chain. This reaction frees the polypeptide. The ribosome separates from the mRNA, and the two ribosomal units disassociate. The subunits are ready to attach to the start codon of another message and start translation anew.

Initiation of Transcription in bacteria

1. A detachable protein called a sigma must bind to the polymerase before transcription can begin. The sigma and the RNA polymerase form a holoenzyme that consists of a core enzyme (RNA polymerase), which contains the active site for catalysis, and other required proteins (sigma). When these two are together, RNA polymerase bonds to only specific sections of DNA - promoters (place where transcription starts.) 2. Sigma makes the initial contact with the DNA of the promoter. Sigma's binding to a promoter determines where and in what direction RNA polymerase will start synthesizing RNA 3. Once the holoenzyme is bounded to a promoter for a bacterial gene, the DNA helix is opened by RNA polymerase, creating two separate strands. The template strand is threaded through a channel that leads to the active site inside of the RNA polymerase. NTPs enter a channel in the enzyme and diffuse to the active site. 4. When an incoming NTP pairs with a complementary base on the template strand of the DNA, RNA polymerization begins. The reaction is exergonic and spontaneous.

Wobble Hypothesis

1. Many amino acids are specified by more than one codon 2. Codons for the same amino acid tend to have the same nucleotides at the first and second positions but a different nucleotide at the third position

Overview of translation

1. Once a NTP (ribonucleoside triphosphate) that matches with a base on the DNA template is in place, RNA polymerase cleaves off two phosphates and catalyzes the formation of phosphodiester linkage between the 3' end of the growing mRNA chain and the new ribonucleoside monophosphate. As this 5' 3' matching-and catalysis process continues, an RNA that is complementary to the gene is synthesized.

Elongation and Termination of Transcription in Bacteria:

1. The RNA polymerase begins moving along the DNA template synthesizing RNA during the elongation phase of transcription. In the interior of the enzyme, a group of amino acids forms a rudder to help steer the template and non-template strand channels inside the enzyme. RNA polymerase adds nucleotides to the 3' end of the growing RNA molecule at a rate of about 50 nucleotides per second. A group of projecting amino acids forms a region called the zipper to help separate the newly synthesized RNA from the DNA template. 2. Transcription stops when RNA polymerase transcribes a DNA sequence that functions as a transcripted-termination signal. The bases that make up the termination signal in bacteria are transcribed into a stretch of RNA with an important property: As soon as I is synthesized, this portion of RNA folds back on itself and forms a short double helix that is held together by complementary base pairing (forms the secondary hair structure - hairpin) The hairpin structure disrupts the interaction between RNA polymerase and RNA transcript, resulting in the physical separation of the enzyme and the product.

Four Steps of Splicing.

1. The process begins when small nucleoproteins (snRNPs - protein plus RNA macromolecular machines) bind to the 5' exon-intron boundary, which is marked by the bases GU, and to a key adenine ribonucleotide (A) near the end of the neutron. 2. Once the initial snRNPs are in place, other snRNPs arrive to form a multipart complex called spliceosome. 3. The intron forms a loop plus a single-stranded stem ( a lariat) with the adenine at its connection point. 4. The lariat is cut out, and a phosphodiester linkage links the exons on either side, producing a continuous coding sequence - mRNA

About how many tRNAs are there?

40. How can all 61 codons be translated with 40 tRNAs? - Wobble Hypothesis

The direction of synthesis of an RNA transcript is _____.

5' —> 3'

Taking into account the wobble hypothesis, which of the following mRNA sequences could be bound to the tRNA anticodon 3' GGC 5'? 3′ CCG 5′ 5′ CCA 3′ 3′ UGC 5′ 5′ UGC 3′

5′ CCA 3′

A particular eukaryotic protein is 300 amino acids long. Which of the following could be the maximum number of nucleotides in the DNA that codes for the amino acid in this protein?

900 It takes 3 nucleotides to code for 3 amino acids, so if there are 300 amino acids then there are 900 nucleotides that are used.

TATA-binding protein

A protein that binds to the TATA box in eukaryotic promoters and is a component of the basal transcription complex

TATA box

A short DNA sequence in many eukaryotic promoters about 30 base pairs upstream from the transcription start site.

promoter

A short nucleotide sequence in DNA that binds to a sigma (in bacteria) or basal transcription factor (in eukaryotes) to enable RNA polymerase to begin transcription In bacteria, several genes are usually are often transcribed from a single promoter. In eukaryotes, each gene generally has its own promoter.

At which site do new aminoacyl tRNAs enter the ribosome during elongation?

A site

start codon

AUG - Methionine -> codon that starts protein synthesis

frameshift mutation

Addition or deletion of a nucleotide reading frame is shifted, altering the meaning of all subsequent codons - almost always deleterious

knock-out, null, loss-of-function allele

Alleles that do not function. Creating a knock-out mutant allele and analyzing their effects is one of the most common research strategies in studies of gene function

tRNA

An RNA that has an anticodon at one end (three ribonucleotides that form base pairs with the mRNA codon) and an amino acid at the other. Each tRNA carries a specific amino acid and binds to the corresponding codon in mRNA during translation

genetic screen

Any technique that identifies individuals with a particular mutation in a poll of a lot of individuals with random mutations.

mutations that are deleterious

Because organisms tend to be well adapted to their current habitat, and because mutations are random changes to the genotype, many mutations lower fitness and are termed harmful or deleterious.

Why did researchers suspect that DNA does not code for proteins directly?

Because there is no chemical complementarity between nucleotides and amino acids; and because in eukaryotes, DNA is in the nucleus but translation occurs in the cytoplasm.

missense mution

Change in nucleotide sequence that changes the amino acid specified by the codon Change in primary structure of protein - may be beneficial, neutral, or deleterious

silent mutation

Change in nucleotide sequence that does not change the amino acid specified by a codon No change in phenotype; neutral with respect to fitness

nonsense mutation

Change in nucleotide sequence that results in an early stop codon Leads to mRNA breakdown or a shortened polypeptide; usually deleterious

Role of Transcription

DNA is transcribed to RNA by RNA polymerase. Transcription is the process of copying hereditary information in DNA to RNA

Alleles of a gene differ in their ______

DNA sequence. As a result, the proteins produced by different alleles of gene may differ in their amino acid sequence.

If a DNA sequence is altered from TAGCTGA to TAGTGA, what kind of mutation has occurred?

Deletion. (frameshift)

True or false? A codon is a group of three bases that can specify more than one amino acid.

False A codon is a group of three bases that can specify only one amino acid.

mutations that are neutral

If a mutation has no effects on fitness, it is termed neutral.

KE Family - FOXP2 gene

In 1990, scientists became interested in the KE family in London, half of whose family members have speech disorders. Analysis of KE family DNA sequences identified a single nucleotide mutation in the FOXP2 gene located on chromosome 7 region 7q31 which correlated with the speech disorder. The defect on the gene is autosomal recessive

Location of translation

In a ribosome in the cytoplasm. Ribosomes contain many proteins and ribosomal RNAs (rRNAs). Ribosomes can be separated into two substructures, called large subunit and small subunit. Each ribosome subunit consists of complex of RNA molecules and proteins. The small subunit holds the mRNA in place during translation; the large subunit is where the peptide-bond formation takes place.

Translation in Bacteria

In bacteria, transcription and translation are tightly coupled. Ribosomes attach to already mature mRNA and begin synthesizing proteins even before transcription is complete. Multiple ribosomes attach to each mRNA, forming a polyribosome - this way many copies of a protein can be produced from a single mRNA. Transcription and translation can occur concurrently in bacteria because there is no nuclear envelope to separate the two processes.

spliceosome

In eukaryotes, a large, complex assembly of snRNPs tat catalyzes removal of introns from primary RNA transcripts.

poly(A) tail

In eukaryotes, a sequence of about 100-250 adeine nucleotides added to the 3' end of the newly transcribed messenger RNA molecules.

poly(A) signal

In eukaryotes, a short sequence of nucleotides near the 3' en of pre-mRNAs that signals cleavage of the RNA and addition of the poly(A) tail

pre-mRNA

In eukaryotes, the primary transcript of protein-ciding genes. Pre-mRNA is processed to form mRNA during RNA Processing

Non-template strand/coding strand

Its sequence matches the sequence of the RNA that is transcribed from the template strand and codes for a polypeptide.

Exceptions to Central Dogma

Many genes code for RNA molecules that do not function as mRNAs - they are not translated into proteins In some cases, information flows from RNA back to DNA. (Some viruses have genes consisting of RNA. So when these RNA viruses infect a cell, a specialized viral polymerase called reverse transcriptase synthesizes a DNA version of the RNA genes that the virus possesses. ex. HIV)

Role of Translation

Messenger RNA is translated into proteins in the ribosomes. Translation is the process of using the information in nucleic acids to synthesize proteins.

upstream

Opposite to the direction in which RNA polymerase moves along a DNA strand

DNA does not store the information to synthesize which of the following? DNA Organelles Proteins Messenger RNA

Organelles

Post Translation Modifications

Proteins fold into the proper shape with the help of molecular chaperones In some cases proteins receive a sugar-based sorting signal that serves as an address label and ensures that the molecule will be carried to the correct location in cell. Sometimes enzymes will remove or add a phosphate group to the protein

mutations that are beneficial

Some mutation increase fitness of the organism - meaning its ability to survive and reproduce - in certain environments.

downstream

The direction in which RNA polymerase moves along a DNA strand (5'-->3')

codon

The group of three bases that specifies a particular amino acid. There are as many as 64 codons that are possible - some specify the same amino acid.

What is meant by translocation?

The ribosome slides one codon down the mRNA.

Central Dogma

The scheme for information flow in the cell: DNA -> RNA --> Protein

Template Strand

The strand that is read by the enzyme (the strand that the RNA builds on)

Francois Jacob and Jacques Monod suggested that RNA molecules act as

a link between genes and the protein-manufacturing centers. They predicted that short lived molecules of RNA (messenger RNA - mRNA) carried information out of the nucleus from DNA to the site of protein synthesis (ribosomes)

metabolic pathway

a linked series of biochemical reactions that build up or breakdown a particular molecule. the product of one reaction is the substrate of the next reaction

During RNA processing a(n) _____ is added to the 3' end of the RNA.

a long string of adenine nucleotides (poly(A) tail)

point mutation

a single-base change -silent -missense -nonsense -frameshift

Transcription begins at a promoter. What is a promoter? a. A site in DNA that recruits the RNA Polymerase b. Part of the RNA molecule itself c. The same as a start codon d. A site found on the RNA polymerase

a. A site in DNA that recruits the RNA Polymerase

What happens to RNA polymerase II after it has completed transcription of a gene? a. It is free to bind to another promoter and begin transcription. b. It is degraded. c. It joins with another RNA polymerase to carry out transcription. d. It begins transcribing the next gene on the chromosome.

a. It is free to bind to another promoter and begin transcription.

Which mutation(s) would not change the remainder of the reading frame of a gene sequence that follows the mutation(s)? a. One addition and one deletion mutation. b. One deletion mutation. c. One addition and two deletion mutations. d. One addition mutation.

a. One addition and one deletion mutation.

Elongation of Translation: Extending the polypeptide

and A sites in the ribosome are empty of tRNAs. As a result an mRNA codon is exposed in the A site. An aminoacyl tRNA binds to the codon in the A site by complementary base paring between the anticodon on the tRNA and the codon on the mRNA. - When both the P site and A site are occupied by tRNAs, the amino acids on the tRNAs are in the ribosome's active site. This is where peptide-bond formation occurs. - When the peptide-bond formation is complete, the polypeptide chain is transferred from the tRNA in the P site to the amino acid held by the tRNA in the A site. - Translocation - Proteins called elongation factors help move ribosome relative to the mRNA so that translation occurs in the 5' 3' direction. Translocation is an energy-demanding event that requires GTP. Translocation moves uncharged RNA into the E site; it moves the tRNA containing the growing polypeptide into the P site; and it opens the A site and exposes a new mRNA codon. The empty tRNA that finds itself in the E site is ejected into the cytosol.

Chromosome of cancer cells exhibit deleterious mutations that include...

aneuploidy, inversions, translocations, deletions, and duplications

Which of the following terms best describes the relationship between the newly synthesized RNA molecule and the DNA template strand? a. Permanently base-paired b. Complementary c. Identical d. Covalently bound

b. Complementary hydrogen bonding

A new type of antibiotic was recently discovered in a fungus native to Hawaii. The substance is a protein that binds specifically to bacterial ribosomes, blocking their function. Why do you think this protein is toxic to bacteria? a. It interferes with mRNA splicing. b. It blocks translation of important mRNAs. c. It blocks transcription of important genes. d. It interferes with RNA polymerase activity.

b. It blocks translation of important mRNAs.

Which of the following mutations would be most likely to have a harmful effect on an organism? a. a deletion of three nucleotides near the middle of a gene b. a single nucleotide insertion downstream of, and close to, the start of the coding sequence c. a single nucleotide deletion near the end of the coding sequence d. a base-pair substitution e. a single nucleotide deletion in the middle of an intron

b. a single nucleotide insertion downstream of, and close to, the start of the coding sequence

Which of the following processes is an example of a post-translational modification? a. Elongation b. Peptide bond formation c. Phosphorylation d. Initiation

c. Phosphorylation

Which is the best description of the loops in a tRNA molecule? a. Single-stranded DNA bases b. Regions provided for binding to the mRNA codon c. Regions of the molecules that remain as single-stranded RNA due to the absence of complementary base pairs d. Regions created as the result of hydrogen bonding between base pairs

c. Regions of the molecules that remain as single-stranded RNA due to the absence of complementary base pairs

Generally speaking, which of the following mutations would most severely affect the protein coded for by a gene? a. a base substitution at the beginning of the gene b. a base substitution at the end of the gene c. a frameshift deletion at the beginning of the gene d. a frameshift deletion at the end of the gene

c. a frameshift deletion at the beginning of the gene

stop (termination) codon

codon that stops/terminates protein synthesis UAA, UAG, UGA - don't code for any amino acid

The anticodon of a particular tRNA molecule is

complementary to the corresponding mRNA codon.

Location? Attachment of amino acid to tRNA

cytoplasm

Translation occurs in the _____.

cytoplasm

Which of the following statements about mutations is false? a. An addition mutation results in an added base in the DNA sequence. b. Addition and deletion mutations disrupt the primary structure of proteins. c. A deletion mutation results in the loss of a base in the DNA sequence. d. A knock-out mutation results in a total absence of the mutated protein.

d. A knock-out mutation results in a total absence of the mutated protein. A knock-out mutation refers to the loss of a protein's function but not necessarily to its complete absence.

Which of the following is not true of RNA processing? a. RNA splicing can be catalyzed by spliceosomes. b. Ribozymes may function in RNA splicing. c. Nucleotides may be added at both ends of the RNA. d. Exons are cut out before mRNA leaves the nucleus. e. A primary transcript is often much longer than the final RNA molecule that leaves the nucleus.

d. Exons are cut out before mRNA leaves the nucleus.

Which of the following steps occurs last in the initiation phase of translation? a. A peptide bond is formed between two adjacent amino acids. b. An aminoacyl tRNA binds to the start codon. c. The small subunit of the ribosome binds to the 5' cap on the mRNA. d. The large ribosomal subunit joins the complex.

d. The large ribosomal subunit joins the complex.

Which of the following statements best describes the promoter of a protein-coding gene? a. The promoter is part of the RNA molecule itself. b. The promoter is a site at which only RNA polymerase will bind. c. The promoter is a site found on RNA polymerase. d.The promoter is a nontranscribed region of a gene.

d.The promoter is a nontranscribed region of a gene. The promoter is the regulatory region of a protein-coding gene at which RNA polymerase must bind to initiate transcription—it is not transcribed into the RNA.

The RNA segments joined to one another by spliceosomes are _____.

exons

True or false. A tRNA with an anticodon complementary to the stop codon catalyzes the reaction by which translation is terminated.

false There are no tRNAs complementary to the three stop codons; termination occurs when release factors recognize the stop codon in the A-site and catalyze the release of the polypeptide from the tRNA in the P-site.

An organism's _____ is determined by the sequences of bases in it's DNA, while its ____ is a product of the proteins it produces

genotype phenotype

Why is the tertiary structure of tRNA important?

it maintains a precise physical distance between the anticodon and amino acid.

RNA processing converts the RNA transcript into _____.

mRNA

During RNA processing a(n) _____ is added to the 5' end of the RNA.

modified guanine nucleotide (cap)

Each gene contained the information...

needed to make an enzyme

Location? Formation of ribosomal subunits

nucleolus

Location? Transcription and RNA processing

nucleous

molecular chaperone

proteins that speed up folding of proteins

Location? Translation of cytoplasmic proteins

ribosome in cytoplasm

Location? Translation of secreted proteins

ribosome on the rough ER

Spliceosomes are composed of _____.

snRNPs and other proteins

RNA polymerase

synthesizes RNA molecules according to the information provided by the sequence of bases in a particular stretch of DNA. Does not require a primer to begin connecting ribonucleotides together to produce a strand of RNA.

Match the biological functions with the three main types of RNAs (tRNA, mRNA, rRNA) -contains exons -is the most abundant form of RNA -has amino acids covalently attached -contains and anticodon -is a component of ribosomes -specifies the amino acid sequence for a protein

tRNA: has amino acids covalently attached; contains and anticodon mRNA: contains exons; specifies the amino acid sequence for a protein rRNA: is the most abundant form of RNA; is a component of ribosomes

Where is an amino acid attached to a tRNA?

the 3' end

karyotype

the complete set of chromosomes in a cell

gene expression

the process of converting archived information into molecules that actually do things in the cell.

triplet code

three base code. A three base code provides more than enough words to code for all 20 amino acids.

Sort the types of RNA where they play their first role of protein synthesis: tRNA pre-mRNA rRNA RNA Primers snRNA mRNA

transcription/RNA processing: pre-mRNA, mRNA, snRNPs (in splicosomes also called snRNA) translation: tRNA, rRNA RNA primers are not used in protein synthesis

Translation of a codon is complete when

when a peptide bond forms between the tRNA amino acid and the growing polypeptide chain

Total number of possible codons based on number of bases and number of bases per codon

x= number of bases y = number of bases per codon x^y = number of possible codons

If proteins were composed of only 12 different kids of amino acids, what would be the smallest possible codon size in a genetic system with four different nucleotides

x= number of bases y = number of bases per codon x^y = number of possible codons 4^y = >12 so 4^1 = 4 wrong 4^2 = 16 (smallest amount that is over 12) so 2 should be to codon size.

Transcription in Eukaryotes

• Eukaryotes have three polymerases - RNA Polymerase 1, 2, and 3. RNA polymerase 2 is used to transcribe protein-coding genes. • Promoters in eukaryotic DNA are more diverse than bacteria promoters. Most eukaryotic promoters include a sequence called the TATA box, centered about 30 base pairs upstream of the transcripting start site, and other important sequences that vary more widely • Instead of using a sigma protein, RNA polymerases recognize promoters using a group of proteins called basal transcription factors. Basal transcription factors assemble at the promoter, and RNA polymerase flows • Termination of eukaryotic protein-coding genes involve a short sequence called the polyadenylation signal or a poly(A) signal. Soon after the signal is transcribed, the RNA is cut by an enzyme downstream of the poly(A) signal as the polymerase continues to transcribe the DNA template. Eventually RNA polymerase falls off the DNA template strand and terminates transcription. Transcription ends variable distances from the poly(A) signal.

RNA Processing & Splicing in Eukaryotes (during transcription)

• When eukaryotic genes of any type are transcribed, the initial product is termed a primary transcript. This RNA must undergo multi-step processing before it is functional. For protein coding genes, the primary transcript is called pre-mRNA. • As transcription proceeds, introns (sections of the primary transcript that are not to be in the mature mRNA) are removed from the growing RNA strand by a process known as splicing. Splicing occurs in the nucleus while transcription is still occurring and results in RNA that contained an uninterrupted genetic message. Only the exons (regions of the eukaryotic genes that are a part of the final mRNA) are leftover. • As soon as the 5' end of a eukaryotic pre-mRNA emerges from RNA polymerase, enzymes add a structure called the 5' cap. The cap consists of a modified guanine nucleotide with three phosphate groups. • An enzyme cleaves the 3' end of the pre-mRNA downstream of the poly(A) signal. Another enzyme adds a long row of 100-250 adenine nucleotides that are not encoded on the DNA template strand. This string is called the poly(A) tail. • With the addition of the poly(A) tail and cap and completion of splicing, processing of pre-mRNA is complete = mature mRNA • Mature mRNA contains UTRs - untranslated 5' and 3' regions that help stabilize the mature RNA and regulate its translation. Bacteria also contains UTRs.